Anti-transthyretin antibodies

ABSTRACT

The invention provides antibodies that specifically bind to transthyretin (TTR). The antibodies can be used for treating or effecting prophylaxis of diseases or disorders associated with TTR accumulation or accumulation of TTR deposits (e.g., TTR amyloidosis). The antibodies can also be used for diagnosing TTR amyloidosis and inhibiting or reducing aggregation of TTR, among other applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 15/201,416 filed Jul.2, 2016, which is a continuation in part of Ser. No. 15/009,666 filedJan. 28, 2016, which claims the benefit of U.S. Provisional ApplicationNo. 62/109,001 filed Jan. 28, 2015 and U.S. Provisional Application No.62/266,557 filed Dec. 11, 2015, each of which is incorporated byreference in its entirety.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named508913_SEQLST.txt, created Jan. 3, 2018 and containing 70,850 bytes,which is hereby incorporated by reference in its entirety for allpurposes.

BACKGROUND

Several diseases are thought to be caused by the abnormal folding andaggregation of disease-specific proteins. These proteins can accumulateinto pathologically diagnostic accumulations, known as amyloids, whichare visualized by certain histologic stains. Amyloids are thought toelicit inflammatory responses and have multiple negative consequencesfor the involved tissues. In addition, smaller aggregates of abnormallyfolded protein may exist and exert cytotoxic effects.

Transthyretin (TTR) is one of the many proteins that are known tomisfold and aggregate (e.g., undergo amyloidogenesis).Transthyretin-related amyloidosis encompasses two forms of disease:familial disease arising from misfolding of a mutated or variant TTR,and a sporadic, non-genetic disease caused by misaggregation ofwild-type TTR. The process of TTR amyloidogenesis can cause pathology inthe nervous system and/or heart, as well as in other tissues.

SUMMARY OF THE CLAIMED INVENTION

In one aspect, the invention provides antibodies that specifically bindtransthyretin comprising three heavy chain CDRs and three light chainCDRs substantially from antibody 6C1. Some such antibodies comprisethree Kabat heavy chain CDRs (SEQ ID NOS: 10-12, respectively) and threelight CDRs (SEQ ID NOS: 18-20, respectively) of antibody 6C1. In someantibodies, the heavy chain CDR-H1 is a composite Kabat-Chothia CDR-H1(SEQ ID NO: 63). Some such antibodies are monoclonal antibodies. Somesuch antibodies are chimeric, humanized, veneered, or human antibodies.Some such antibodies have a human IgG1 isotype. Some such antibodieshave a human IgG2 or IgG4 isotype.

Some such antibodies are humanized or chimeric 6C1 antibodies thatspecifically bind to transthyretin, wherein 6C1 is a mouse antibodycharacterized by a mature heavy chain variable region of SEQ ID NO:1 anda mature light chain variable region of SEQ ID NO:13.

In some antibodies, the humanized mature heavy chain variable regioncomprises the three heavy chain CDRs of 6C1 and the humanized maturelight chain variable region comprises the three light chain CDRs of 6C1.In some antibodies, the humanized mature heavy chain variable regioncomprises the three Kabat heavy chain CDRs of 6C1 (SEQ ID NOs:10-12) andthe humanized mature light chain variable region comprises the threeKabat light chain CDRs of 6C1 (SEQ ID NOs:18-20).

In some antibodies, the humanized mature heavy chain variable region hasan amino acid sequence at least 90% identical to SEQ ID NO:9 and thehumanized mature light chain variable region has an amino acid sequenceat least 90% identical to SEQ ID NO:17. In some such antibodies,position H77 is occupied by T. In some such antibodies, position H49 isoccupied by A. In some such antibodies, positions H76 and H82(a) areoccupied by S. In some such antibodies, position H49 is occupied by A.In some such antibodies, positions H19, H44, H83, and H89 are occupiedby K, R, K, and M, respectively. In some such antibodies, position H49is occupied by A. In some such antibodies, position L45 is occupied byK. In some such antibodies, position L2 is occupied by V.

Some antibodies comprise a mature heavy chain variable region having anamino acid sequence at least 95% identical to SEQ ID NO:9 and a maturelight chain variable region having an amino acid sequence at least 95%identical to SEQ ID NO:17. Some antibodies comprise a mature heavy chainvariable region having an amino acid sequence at least 98% identical toSEQ ID NO:9 and a mature light chain variable region having an aminoacid sequence at least 98% identical to SEQ ID NO:17.

In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:4. In some such antibodies, the matureheavy chain variable region has an amino acid sequence of SEQ ID NO:5.In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:6. In some such antibodies, the matureheavy chain variable region has an amino acid sequence of SEQ ID NO:7.In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:8. In some such antibodies, the matureheavy chain variable region has an amino acid sequence of SEQ ID NO:9.

In some such antibodies, the mature light chain variable region has anamino acid sequence of SEQ ID NO:16. In some such antibodies, the maturelight chain variable region has an amino acid sequence of SEQ ID NO:17.

In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:4 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:16. In some suchantibodies, the mature heavy chain variable region has an amino acidsequence of SEQ ID NO:4 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO:17.

In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:5 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:16. In some suchantibodies, the mature heavy chain variable region has an amino acidsequence of SEQ ID NO:5 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO:17.

In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:6 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:16. In some suchantibodies, the mature heavy chain variable region has an amino acidsequence of SEQ ID NO:6 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO:17.

In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:7 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:16. In some suchantibodies, the mature heavy chain variable region has an amino acidsequence of SEQ ID NO:7 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO:17.

In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:8 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:16. In some suchantibodies, the mature heavy chain variable region has an amino acidsequence of SEQ ID NO:8 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO:17.

In some such antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:9 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:16. In some suchantibodies, the mature heavy chain variable region has an amino acidsequence of SEQ ID NO:9 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO:17.

In some antibodies, the antibody is an intact antibody. In someantibodies, the antibody is a binding fragment. In some such antibodies,the binding fragment is a single-chain antibody, Fab, or Fab′2 fragment.

In some antibodies, the mature light chain variable region is fused to alight chain constant region and the mature heavy chain variable regionis fused to a heavy chain constant region. In some such antibodies, theheavy chain constant region is a mutant form of a natural human heavychain constant region which has reduced binding to a Fcγ receptorrelative to the natural human heavy chain constant region. In some suchantibodies, the heavy chain constant region is of IgG1 isotype. In somesuch antibodies, the mature heavy chain variable region is fused to aheavy chain constant region having the sequence of SEQ ID NO:26 and/orthe mature light chain variable region is fused to a light chainconstant region having the sequence of SEQ ID NO:28.

In some antibodies, any differences in CDRs of the mature heavy chainvariable region and mature light chain variable region from SEQ ID NOS:1and 13, respectively, reside in positions H60-H65.

In another aspect, the invention provides a pharmaceutical compositioncomprising the any of the above mentioned antibodies and apharmaceutically acceptable carrier.

In another aspect, the invention provides a nucleic acid encoding theheavy chain and/or light chain of any of the above mentioned antibodies.In another aspect, the invention provides a recombinant expressionvector comprising such a nucleic acid. In another aspect, the inventionprovides a host cell transformed with such a recombinant expressionvector.

In another aspect, the invention provides a method of humanizing anantibody, the method comprising:

-   -   (a) selecting an acceptor antibody;    -   (b) identifying the amino acid residues of the mouse antibody to        be retained;    -   (c) synthesizing a nucleic acid encoding a humanized heavy chain        comprising CDRs of the mouse antibody heavy chain and a nucleic        acid encoding a humanized light chain comprising CDRs of the        mouse antibody light chain; and    -   (d) expressing the nucleic acids in a host cell to produce a        humanized antibody;        wherein the mouse antibody comprises a heavy chain variable        region having an amino acid sequence of SEQ ID NO:1 and a light        chain variable region having an amino acid sequence of SEQ ID        NO:13.

In another aspect, the invention provides a method of producing ahumanized, chimeric, or veneered antibody, the method comprising:

-   -   (a) culturing cells transformed with nucleic acids encoding the        heavy and light chains of the antibody, so that the cells        secrete the antibody; and    -   (b) purifying the antibody from cell culture media;        wherein the antibody is a humanized, chimeric, or veneered form        of 6C1.

In another aspect, the invention provides a method of producing a cellline producing a humanized, chimeric, or veneered antibody, the methodcomprising:

-   -   (a) introducing a vector encoding heavy and light chains of an        antibody and a selectable marker into cells;    -   (b) propagating the cells under conditions to select for cells        having increased copy number of the vector;    -   (c) isolating single cells from the selected cells; and    -   (d) banking cells cloned from a single cell selected based on        yield of antibody;        wherein the antibody is a humanized, chimeric, or veneered form        of 6C1.

Some such methods further comprise propagating the cells under selectiveconditions and screening for cell lines naturally expressing andsecreting at least 100 mg/L/10⁶ cells/24 h.

In another aspect, the invention provides a method of inhibiting orreducing aggregation of transthyretin in a subject having or at risk ofdeveloping a transthyretin-mediated amyloidosis, comprisingadministering to the subject an effective regime of any of the abovementioned antibodies, thereby inhibiting or reducing aggregation oftransthyretin in the subject.

In any of the above methods, the transthyretin-mediated amyloidosis canbe associated with a condition selected from any of cardiomyopathy orhypertrophy, familial amyloid polyneuropathy, central nervous systemselective amyloidosis (CNSA), senile systemic amyloidosis, senilecardiac amyloidosis, spinal stenosis, osteoarthritis, rheumatoidarthritis, juvenile idiopathic arthritis, macular degeneration and aligament or tendon disorder.

The invention further provides a method of treating a subject having orat risk of any of cardiomyopathy or hypertrophy, familial amyloidpolyneuropathy, central nervous system selective amyloidosis (CNSA),senile systemic amyloidosis, senile cardiac amyloidosis, spinalstenosis, osteoarthritis, rheumatoid arthritis, juvenile idiopathicarthritis, macular degeneration and a ligament or tendon disorder, themethod comprising administering to the subject an effective regime ofthe antibody of any one of claims.

In another aspect, the invention provides a method of inhibiting orreducing transthyretin fibril formation in a subject having or at riskof developing a transthyretin-mediated amyloidosis, comprisingadministering to the subject an effective regime of any of the abovementioned antibodies, thereby inhibiting or reducing transthyretinaccumulation in the subject.

In another aspect, the invention provides a method of reducingtransthyretin deposits in a subject having or at risk of developing atransthyretin-mediated amyloidosis, comprising administering to thesubject an effective regime of any of the above mentioned antibodies,thereby reducing transthyretin deposits in the subject.

In another aspect, the invention provides a method of clearingaggregated transthyretin in a subject having or at risk of developing atransthyretin-mediated amyloidosis, comprising administering to thesubject an effective regime of any of the above mentioned antibodies,thereby clearing aggregated transthyretin from the subject relative to asubject having or at risk of developing a transthyretin-mediatedamyloidosis who has not received the antibody.

In another aspect, the invention provides a method of stabilizing anon-toxic conformation of transthyretin in a subject having or at riskof developing a transthyretin-mediated amyloidosis, comprisingadministering to the subject an effective regime of any of the abovementioned antibodies, thereby stabilizing a non-toxic conformation oftransthyretin in the subject.

In another aspect, the invention provides a method of treating oreffecting prophylaxis of a transthyretin-mediated amyloidosis in asubject, comprising administering to the subject an effective regime ofany of the above mentioned antibodies.

In another aspect, the invention provides a method of delaying the onsetof a transthyretin-mediated amyloidosis in a subject, comprisingadministering to the subject an effective regime of any of the abovementioned antibodies.

In another aspect, the invention provides a method of diagnosing atransthyretin-mediated amyloidosis in a subject, comprising contacting abiological sample from the subject with an effective amount of any ofthe above mentioned antibodies. Some such methods further comprisedetecting the binding of antibody to transthyretin, wherein the presenceof bound antibody indicates the subject has a transthyretin-mediatedamyloidosis. Some such methods further comprise comparing binding of theantibody to the biological sample with binding of the antibody to acontrol sample, whereby increased binding of the antibody to thebiological sample relative to the control sample indicates the subjecthas a transthyretin-mediated amyloidosis.

In some such methods, the biological sample and the control samplecomprise cells of the same tissue origin. In some such methods, thebiological sample and/or the control sample is blood, serum, plasma, orsolid tissue. In some such methods, the solid tissue is from the heart,peripheral nervous system, autonomic nervous system, kidneys, eyes, orgastrointestinal tract.

In some methods, the transthyretin-mediated amyloidosis is a familialtransthyretin amyloidosis or a sporadic transthyretin amyloidosis. Insome such methods, the familial transthyretin amyloidosis is familialamyloid cardiomyopathy (FAC), familial amyloid polyneuropathy (FAP), orcentral nervous system selective amyloidosis (CNSA). In some suchmethods, the sporadic transthyretin amyloidosis is senile systemicamyloidosis (SSA) or senile cardiac amyloidosis (SCA).

In some methods, the transthyretin-mediated amyloidosis is associatedwith amyloid accumulation in the heart, peripheral nervous system,autonomic nervous system, kidneys, eyes, or gastrointestinal tract ofthe subject.

In another aspect, the invention provides a method of detecting thepresence or absence of transthyretin deposits in a subject, comprisingcontacting a biological sample from the subject suspected of comprisingthe amyloid accumulation with an effective amount of any of the abovementioned antibodies. Some such methods further comprise detecting thebinding of antibody to transthyretin, wherein detection of boundantibody indicates the presence of transthyretin deposits. Some suchmethods further comprise comparing binding of the antibody to thebiological sample with binding of the antibody to a control sample,whereby increased binding of the antibody to the biological samplerelative to the control sample indicates the subject has atransthyretin-mediated amyloidosis. In some such methods, the biologicalsample and the control sample comprise cells of the same tissue origin.In some such methods, the biological sample and/or the control sample isblood, serum, plasma, or solid tissue. In some such methods, the solidtissue is from the heart, peripheral nervous system, autonomic nervoussystem, kidneys, eyes, or gastrointestinal tract.

In another aspect, the invention provides a method of determining alevel of transthyretin deposits in a subject, comprising administeringany of the above mentioned antibodies and detecting the presence ofbound antibody in the subject. In some such methods, the presence ofbound antibody is determined by positron emission tomography (PET).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent application with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts an alignment of heavy chain variable regions of the mouse6C1 antibody, mouse model antibodies, human acceptor antibodies, andhumanized versions of the 6C1 antibody. The CDRs as defined by Kabat areenclosed in boxes, except that the first enclosed box is a composite ofthe Chothia CDR-H1 and the Kabat CDR-H1, with the Kabat CDR-H1underlined and bolded.

FIG. 2 depicts an alignment of light chain variable regions of the mouse6C1 antibody, mouse model antibodies, human acceptor antibodies, andhumanized versions of the 6C1 antibody. The CDRs as defined by Kabat areenclosed in boxes.

FIGS. 3A & 3B: FIG. 3A depicts the binding curve of murine 5A1, 6C1,9D5, and 14G8 antibodies to ph4-treated TTR. FIG. 3B depicts the bindingcurve of murine 5A1, 6C1, 9D5, and 14G8 antibodies to ph4-treated ornative TTR

FIG. 4A, 4B & 4C: FIG. 4A depicts the inhibition of TTR-Y78F fiberformation by mis-TTR antibodies. FIG. 4B depicts the inhibition ofTTR-V122I fiber formation by 14G8.

FIG. 4C depicts the inhibition of TTR-V122I fiber formation by a controlantibody.

FIGS. 5A & 5B: FIG. 5A depicts a densitometry analysis of a Western Blotanalysis of plasma samples from patients confirmed for V30M ATTR (Sample#11, #12, #15, #18, #19, ##20) and samples from normal subjects (Sample#21, #22, #23, #24, #25, and #27) using the 9D5 mis-TTR antibody. FIG.5B depicts a densitometry analysis of a Western blot analysis of thesame samples using the 5A1 mis-TTR antibody.

FIG. 6 depicts a MesoScale Discovery (MSD) plate assay of plasma samplesfrom patients confirmed for V30M ATTR (Sample #11, #12, #15, #18, #19,#20) and samples from normal subjects (#21, #22, #23, #24, #25, #27)using the 6C1 antibody.

FIGS. 7A & 7B: FIG. 7A depicts the effect of antibody 14G8 on the uptakeof F87M/L110M TTR by THP-1 cells. FIG. 7B depicts the effect of each ofthe mis-TTR antibodies on the uptake of V30M TTR by THP-1 cells.

FIGS. 8A-C and FIG. 8D. 1-2 14G8 binds to TTR-V122I fibril ends and tooligomeric aggregates as assessed using TEM and AFM. Immunogold labelingwith 14G8 was observed in TTR-V122I oligomer aggregates and fibril ends(FIG. 8A), whereas immunogold labeling with an anti-TTR pAb showedbinding along the lengths of TTR fibers and to oligomeric clusters (FIG.8B). IgG1 isotype control mAb did not show immunogold labeling (FIG.8C). TTR-V122I fibers, alone and in the presence of 14G8±6 nm colloidalgold-conjugated secondary antibody, were assessed using AFM. Goldlabeling was observed at fiber ends (FIG. 8D)

FIGS. 9A. 1-4 and 9B: Interaction of 14G8 with mature TTR-V122I fibrilsassessed using ITC fits to a 2-binding site model. ITC data and bindingisotherms for 14G8 binding to aggregated TTR variants are presented inFIG. 8A. Binding was fit to a 2-binding site model with KD values shown(FIG. 8B).

FIG. 10A. 1-2, 10B. 1-2, 10C. 1-2, 10D-F, and 10G. 1-3 show 14G8immunolabeled TTR amyloid present between fibers of the nerve fascicle apatient with ATTR amyloidosis resulting from a TTR-V30M mutation. FIG.10A panels 1 and 2 show amyloid between fibers of the nerve fascicle,which overlapped with staining by Congo red (FIG. 8B panels 1 and 2) andthioflavin T (FIG. 10C panels 1 and 2), and immunolabeling by atotal-TTR antibody (FIG. 10D) in tissue derived from a patient with ATTRamyloidosis. No staining was seen with the use of 2 isotype controlantibodies (FIGS. 10E-F); however, axonal degeneration (lack of Schwanncell nuclei) in the areas laden with TTR amyloid deposits were alsoobserved (FIGS. 10E-F [red areas in 6E]). Peripheral nerves from ahealthy control were not labeled using either 14G8 or a total-TTRantibody (FIG. 10G panels 1-3).

FIG. 11A. 1-3, 11B. 1-3, 11C. 1-3, 11D. 1-3 and 11E. 1-4 shows antibody14G8 immunolabels TTR amyloid in the gastrointestinal tract derived froma patient with TTR-C30M amyloidosis. FIGS. 11A, B panels 1 showMeissner's plexus and glands in the esophagus, FIG. 11C panel 1 showsthe rich vasculature bed in the submucosa, FIG. 11D panel 1 shows themuscularis propria (MP) and muscularis mucosa 14G8-positive TTR amyloidoverlapped with Congo red fluorescent staining (FIGS. 11A-D panels 2).FIGS. 11A-D panels 3 show ATTR amyloidosis tissue stained with anisotype control mAb 14G8 immunoreactivity was absent in healthy controltissue (FIG. 11 panels 1-4).

FIGS. 12A-12D show exemplary humanized Vh designs, with backmutationsand other mutations based on selected human frameworks The gray-shadedareas in the first column indicate the CDRs as defined by Chothia, andthe gray-shaded areas in the remaining columns indicate the CDRs asdefined by Kabat.

FIGS. 13A-13D show exemplary humanized Vk designs, with backmutationsand other mutations based on selected human frameworks. The gray-shadedareas in the first column indicate the CDRs as defined by Chothia, andthe gray-shaded areas in the remaining columns indicate the CDRs asdefined by Kabat.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 6C1 antibody.

SEQ ID NO:2 sets forth the amino acid sequence of the mouse heavy chainvariable region structure template.

SEQ ID NO:3 sets forth the amino acid sequence of the heavy chainvariable acceptor accession number ADX65650.

SEQ ID NO:4 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 6C1 antibody version 1 (Hu6C1VHv1).

SEQ ID NO:5 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 6C1 antibody version 1b (Hu6C1VHv1b).

SEQ ID NO:6 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 6C1 antibody version 2 (Hu6C1VHv2).

SEQ ID NO:7 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 6C1 antibody version 2b (Hu6C1VHv2b).

SEQ ID NO:8 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 6C1 antibody version 3 (Hu6C1VHv3).

SEQ ID NO:9 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 6C1 antibody version 3b (Hu6C1VHv3b).

SEQ ID NO:10 sets forth the amino acid sequence of Kabat CDR-H1 of themouse 6C1 antibody.

SEQ ID NO:11 sets forth the amino acid sequence of Kabat CDR-H2 of themouse 6C1 antibody.

SEQ ID NO:12 sets forth the amino acid sequence of Kabat CDR-H3 of themouse 6C1 antibody.

SEQ ID NO:13 sets forth the amino acid sequence of the light chainvariable region of the mouse 6C1 antibody.

SEQ ID NO:14 sets forth the amino acid sequence of the mouse light chainvariable region structure template.

SEQ ID NO:15 sets forth the amino acid sequence of the light chainvariable acceptor accession number ABI74084.

SEQ ID NO:16 sets forth the amino acid sequence of the light chainvariable region of the humanized 6C1 antibody version 1 (Hu6C1VLv1).

SEQ ID NO:17 sets forth the amino acid sequence of the light chainvariable region of the humanized 6C1 antibody version 2 (Hu6C1VLv2).

SEQ ID NO:18 sets forth the amino acid sequence of Kabat CDR-L1 of themouse 6C1 antibody.

SEQ ID NO:19 sets forth the amino acid sequence of Kabat CDR-L2 of themouse 6C1 antibody.

SEQ ID NO:20 sets forth the amino acid sequence of Kabat CDR-L3 of themouse 6C1 antibody.

SEQ ID NO:21 sets forth a nucleic acid sequence encoding the heavy chainvariable region of the mouse 6C1 antibody with signal peptide.

SEQ ID NO:22 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 6C1 antibody with signal peptide.

SEQ ID NO:23 sets forth a nucleic acid sequence encoding the light chainvariable region of the mouse 6C1 antibody with signal peptide.

SEQ ID NO:24 sets forth the amino acid sequence of the light chainvariable region of the mouse 6C1 antibody with signal peptide.

SEQ ID NO:25 sets forth the amino acid sequence of an exemplary IgG1heavy chain constant region.

SEQ ID NO:26 sets forth the amino acid sequence of an exemplary IgG1G1m3 heavy chain constant region.

SEQ ID NO:27 sets forth the amino acid sequence of an exemplary IgG1G1m3 heavy chain constant region.

SEQ ID NO:28 sets forth the amino acid sequence of an exemplary lightchain constant region with C-terminal Arginine.

SEQ ID NO:29 sets forth the amino acid sequence of an exemplary lightchain constant region without C-terminal Arginine.

SEQ ID NO:30 sets forth the amino acid sequence of the heavy chainregion of the humanized 6C1 antibody version 1.

SEQ ID NO:31 sets forth the amino acid sequence of the heavy chainregion of the humanized 6C1 antibody version 1b.

SEQ ID NO:32 sets forth the amino acid sequence of the heavy chainregion of the humanized 6C1 antibody version 2.

SEQ ID NO:33 sets forth the amino acid sequence of the heavy chainregion of the humanized 6C1 antibody version 2b.

SEQ ID NO:34 sets forth the amino acid sequence of the heavy chainregion of the humanized 6C1 antibody version 3.

SEQ ID NO:35 sets forth the amino acid sequence of the heavy chainregion of the humanized 6C1 antibody version 3b.

SEQ ID NO:36 sets forth the amino acid sequence of the light chainregion of the humanized 6C1 antibody version 1.

SEQ ID NO:37 sets forth the amino acid sequence of the light chainregion of the humanized 6C1 antibody version 2.

SEQ ID NO:38 sets forth the amino acid sequence of human transthyretinset forth in accession number P02766.1 (UniProt).

SEQ ID NO:39 sets forth the amino acid sequence of human transthyretinset forth in accession number AAB35639.1 (GenBank).

SEQ ID NO:40 sets forth the amino acid sequence of human transthyretinset forth in accession number AAB35640.1 (GenBank).

SEQ ID NO:41 sets forth the amino acid sequence of human transthyretinset forth in accession number and ABI63351.1 (GenBank).

SEQ ID NO:42 sets forth the amino acid sequence of residues 89-97 ofhuman transthyretin.

SEQ ID NO:43 sets forth the amino acid sequence of a potentialtransthyretin immunogen.

SEQ ID NO:44 sets forth the amino acid sequence of a potentialtransthyretin immunogen.

SEQ ID NO:45 sets forth the amino acid sequence of a potentialtransthyretin immunogen.

SEQ ID NO:46 sets forth a nucleic acid sequence encoding an exemplaryIgG1 G1m3 heavy chain constant region.

SEQ ID NO:47 sets forth a nucleic acid sequence encoding an exemplarylight chain constant region with C-terminal Arginine.

SEQ ID NO:48 sets forth a nucleic acid sequence encoding an exemplarylight chain constant region without C-terminal Arginine.

SEQ ID NO:49 sets forth the amino acid sequence of a heavy chainconstant region signal peptide.

SEQ ID NO:50 sets forth a nucleic acid sequence encoding a heavy chainconstant region signal peptide.

SEQ ID NO:51 sets forth the amino acid sequence of a light chainconstant region signal peptide.

SEQ ID NO:52 sets forth a nucleic acid sequence encoding a light chainconstant region signal peptide.

SEQ ID NO:53 sets forth a nucleic acid sequence encoding a mouse 6C1variable light chain region.

SEQ ID NO:54 sets forth a nucleic acid sequence encoding a mouse 6C1variable heavy chain region.

SEQ ID NO:55 sets forth a nucleic acid sequence encoding a heavy chainvariable region of the humanized 6C1 antibody version 1 (Hu6C1VHv1).

SEQ ID NO:56 sets forth a nucleic acid sequence encoding a heavy chainvariable region of the humanized 6C1 antibody version 1b (Hu6C1VHv1b).

SEQ ID NO:57 sets forth a nucleic acid sequence encoding a heavy chainvariable region of the humanized 6C1 antibody version 2 (Hu6C1VHv2).

SEQ ID NO:58 sets forth a nucleic acid sequence encoding a heavy chainvariable region of the humanized 6C1 antibody version 2b (Hu6C1VHv2b).

SEQ ID NO:59 sets forth a nucleic acid sequence encoding a heavy chainvariable region of the humanized 6C1 antibody version 3 (Hu6C1VHv3).

SEQ ID NO:60 sets forth a nucleic acid sequence encoding a heavy chainvariable region of the humanized 6C1 antibody version 3b (Hu6C1VHv3b).

SEQ ID NO:61 sets forth a nucleic acid sequence encoding a light chainvariable region of the humanized 6C1 antibody version 1 (Hu6C1VLv1).

SEQ ID NO:62 sets forth a nucleic acid sequence encoding a light chainvariable region of the humanized 6C1 antibody version 2 (Hu6C1VLv2).

SEQ ID NO:63 sets forth the amino acid sequence of a composite CDR-H1(residues 26-35) of the mouse 6C1 antibody.

Definitions

Monoclonal antibodies or other biological entities are typicallyprovided in isolated form. This means that an antibody or otherbiologically entity is typically at least 50% w/w pure of interferingproteins and other contaminants arising from its production orpurification but does not exclude the possibility that the monoclonalantibody is combined with an excess of pharmaceutically acceptablecarrier(s) or other vehicle intended to facilitate its use. Sometimesmonoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/wpure of interfering proteins and contaminants from production orpurification. Often an isolated monoclonal antibody or other biologicalentity is the predominant macromolecular species remaining after itspurification.

Specific binding of an antibody to its target antigen means an affinityof at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific binding isdetectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces. Specific bindingdoes not however necessarily imply that an antibody binds one and onlyone target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. See generally,Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7 (incorporated by reference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region (also referred toherein as a “light chain variable domain” (“VL domain”) or “heavy chainvariable domain” (“VH domain”), respectively) consists of a “framework”region interrupted by three “complementarity determining regions” or“CDRs.” The framework regions serve to align the CDRs for specificbinding to an epitope of an antigen. The CDRs include the amino acidresidues of an antibody that are primarily responsible for antigenbinding. From amino-terminus to carboxyl-terminus, both VL and VHdomains comprise the following framework (FR) and CDR regions: FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain arealso referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3;CDRs 1, 2, and 3 of a VH domain are also referred to herein,respectively, as CDR-H1, CDR-H2, and CDR-H3.

The assignment of amino acids to each VL and VH domain is in accordancewith any conventional definition of CDRs. Conventional definitionsinclude, the Kabat definition (Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), The Chothia definition (Chothia & Lesk, J. Mol. Biol.196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); acomposite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothiaand Kabat CDRs; the AbM definition used by Oxford Molecular's antibodymodelling software; and, the contact definition of Martin et al(bioinfo.org.uk/abs) (see Table 1). Kabat provides a widely usednumbering convention (Kabat numbering) in which corresponding residuesbetween different heavy chains or between different light chains areassigned the same number. When an antibody is said to comprise CDRs by acertain definition of CDRs (e.g., Kabat) that definition specifies theminimum number of CDR residues present in the antibody (i.e., the KabatCDRs). It does not exclude that other residues falling within anotherconventional CDR definition but outside the specified definition arealso present. For example, an antibody comprising CDRs defined by Kabatincludes among other possibilities, an antibody in which the CDRscontain Kabat CDR residues and no other CDR residues, and an antibody inwhich CDR H1 is a composite Chothia-Kabat CDR H1 and other CDRs containKabat CDR residues and no additional CDR residues based on otherdefinitions.

TABLE 1 Conventional Definitions of CDRs Using Kabat Numbering Compositeof Loop Kabat Chothia Chothia & Kabat AbM Contact L1 L24--L34 L24--L34L24--L34 L24--L34 L30--L36 L2 L50--L56 L50--L56 L50--L56 L50--L56L46--L55 L3 L89--L97 L89--L97 L89--L97 L89--L97 L89--L96 H1 H31--H35BH26--H32..H34* H26--H35B* H26--H35B H30--H35B H2 H50--H65 H52--H56H50--H65 H50--H58 H47--H58 H3 H95--H102 H95--H102 H95--H102 H95--H102H93--H101 *CDR-H1 by Chothia can end at H32, H33, or H34 (depending onthe length of the loop). This is because the Kabat numbering schemeplaces insertions of extra residues at 35A and 35B, whereas Chothianumbering places them at 31A and 31B. If neither H35A nor H35B (Kabatnumbering) is present, the Chothia CDR-H1 loop ends at H32. If only H35Ais present, it ends at H33. If both H35A and H35B are present, it endsat H34.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target includingseparate heavy chains, light chains Fab, Fab′, F(ab′)2, F(ab)c, Dabs,nanobodies, and Fv. Fragments can be produced by recombinant DNAtechniques, or by enzymatic or chemical separation of intactimmunoglobulins. The term “antibody” also includes a bispecific antibodyand/or a humanized antibody. A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites (see, e.g., Songsivilai and Lachmann,Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol.,148:1547-53 (1992)). In some bispecific antibodies, the two differentheavy/light chain pairs include a humanized 6C1 heavy chain/light chainpair and a heavy chain/light chain pair specific for a different epitopeon transthyretin than that bound by 6C1.

In some bispecific antibodies, one heavy chain/light chain pair is ahumanized 6C1 antibody as further disclosed below and the other heavychain/light chain pair is from an antibody that binds to a receptorexpressed on the blood brain barrier, such as an insulin receptor, aninsulin-like growth factor (IGF) receptor, a leptin receptor, or alipoprotein receptor, or a transferrin receptor (Friden et al., Proc.Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science259:373-377, 1993). Such a bispecific antibody can be transferred crossthe blood brain barrier by receptor-mediated transcytosis. Brain uptakeof the bispecific antibody can be further enhanced by engineering thebi-specific antibody to reduce its affinity to the blood brain barrierreceptor. Reduced affinity for the receptor resulted in a broaderdistribution in the brain (see, e.g., Atwal et al., Sci. Trans. Med. 3,84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).

Exemplary bispecific antibodies can also be: (1) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg(2010)); (2) a Tandab, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (3) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(4) a so-called “dock and lock” molecule, based on the “dimerization anddocking domain” in Protein Kinase A, which, when applied to Fabs, canyield a trivalent bispecific binding protein consisting of two identicalFab fragments linked to a different Fab fragment; or (5) a so-calledScorpion molecule, comprising, e.g., two scFvs fused to both termini ofa human Fc-region. Examples of platforms useful for preparing bispecificantibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2(F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab armexchange, Genmab).

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids (also known aslinear epitopes) are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding (also known asconformational epitopes) are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996). The epitope can be linear, such as anepitope of, for example, 2-5, 3-5, 3-9, or 5-9 contiguous amino acidsfrom SEQ ID NO:38. The epitope can also be a conformational epitopeincluding, for example, two or more non-contiguous segments of aminoacids within residues 89-97 of SEQ ID NO:38. If an antibody is said tobind to an epitope within amino acids 89-97 of transthyretin (TTR), forexample, what is meant is that the epitope is within the recited rangeof amino acids including those defining the outer-limits of the range.It does not necessarily mean that every amino acid within the rangeconstitutes part of the epitope. Thus, for example, an epitope withinamino acids 89-97 of TTR may consist of amino acids 89-97, 89-96, 90-96,91-96, 92-96, 93-96, 94-96, 89-96, 89-95, 89-94, 89-93, 89-92 or 89-93,among other linear segments of SEQ ID NO:42, or in the case ofconformational epitopes, non-contiguous segments of amino acids of SEQID NO:42.

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined X-ray crystallography of theantibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% as measured in acompetitive binding assay. Some test antibodies inhibit binding of thereferences antibody by at least 75%, 90% or 99%. Antibodies identifiedby competition assay (competing antibodies) include antibodies bindingto the same epitope as the reference antibody and antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

The term “native” with respect to the structure transthyretin (TTR)refers to the normal folded structure of TTR in its properly functioningstate (i.e., a TTR tetramer). As TTR is a tetramer in its nativelyfolded form, non-native forms of TTR include, for example, misfolded TTRtetramers, TTR monomers, aggregated forms of TTR, and fibril forms ofTTR. Non-native forms of TTR can include molecules comprising wild-typeTTR amino acid sequences or mutations.

The term “misfolded” with respect to TTR refers to the secondary andtertiary structure of a TTR polypeptide monomer or multimer, andindicates that the polypeptide has adopted a conformation that is notnormal for that protein in its properly functioning state. Although TTRmisfolding can be caused by mutations in the protein (e.g., deletion,substitution, or addition), wild-type TTR proteins can also be misfoldedin diseases, exposing specific epitopes.

The term “pharmaceutically acceptable” means that the carrier, diluent,excipient, or auxiliary is compatible with the other ingredients of theformulation and not substantially deleterious to the recipient thereof.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,and situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “biological sample” refers to a sample of biological materialwithin or obtainable from a biological source, for example a human ormammalian subject. Such samples can be organs, organelles, tissues,sections of tissues, bodily fluids, peripheral blood, blood plasma,blood serum, cells, molecules such as proteins and peptides, and anyparts or combinations derived therefrom. The term biological sample canalso encompass any material derived by processing the sample. Derivedmaterial can include cells or their progeny. Processing of thebiological sample may involve one or more of filtration, distillation,extraction, concentration, fixation, inactivation of interferingcomponents, and the like.

The term “control sample” refers to a biological sample not known orsuspected to include monomeric, misfolded, aggregated, or fibril formsof transthyretin (TTR), such as in TTR amyloid deposits. Control samplescan be obtained from individuals not afflicted with a TTR amyloidosis ora specifically chosen type of TTR amyloidosis. Alternatively, controlsamples can be obtained from patients afflicted with TTR amyloidosis ora specifically chosen type of TTR amyloidosis. Such samples can beobtained at the same time as a biological sample thought to comprise theTTR amyloidosis or on a different occasion. A biological sample and acontrol sample can both be obtained from the same tissue (e.g., a tissuesection containing both TTR amyloid deposits and surrounding normaltissue). Preferably, control samples consist essentially or entirely oftissue free of TTR amyloid deposits and can be used in comparison to abiological sample thought to comprise TTR amyloid deposits. Preferably,the tissue in the control sample is the same type as the tissue in thebiological sample (e.g., cardiomyocytes in the heart).

The term “disease” refers to any abnormal condition that impairsphysiological function. The term is used broadly to encompass anydisorder, illness, abnormality, pathology, sickness, condition, orsyndrome in which physiological function is impaired, irrespective ofthe nature of the etiology.

The term “symptom” refers to a subjective evidence of a disease, such asaltered gait, as perceived by the subject. A “sign” refers to objectiveevidence of a disease as observed by a physician.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (e.g., SEM) of astated value.

Statistical significance means p≤0.05.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

DETAILED DESCRIPTION I. General

The invention provides antibodies that specifically bind to residues89-97 of transthyretin (TTR). The antibodies have the capacity to bindto monomeric, misfolded, aggregated, or fibril forms of TTR. Theantibodies can be used for treating or effecting prophylaxis of diseasesor disorders associated with TTR accumulation or accumulation of TTRdeposits (e.g., TTR amyloidosis). The antibodies can also be used fordiagnosing TTR amyloidosis and inhibiting or reducing aggregation ofTTR, among other applications.

II. Target Molecules

Transthyretin (TTR) is a 127-amino acid, 55 kDa serum and cerebrospinalfluid transport protein primarily synthesized by the liver. It has alsobeen referred to as prealbumin, thyroxine binding prealbumin, ATTR, andTBPA. In its native state, TTR exists as a tetramer. In homozygotes, thetetramers comprise identical 127-amino-acid beta-sheet-rich subunits. Inheterozygotes, the TTR tetramers are made up of variant and/or wild-typesubunits, typically combined in a statistical fashion.

The established function of TTR in the blood is to transportholo-retinol binding protein. Although TTR is the major carrier ofthyroxine (T4) in the blood of rodents, utilizing binding sites that areorthogonal to those used for holo-retinol binding protein, the T4binding sites are effectively unoccupied in humans.

TTR is one of at least thirty different human proteins whoseextracellular misfolding and/or misassembly (amyloidogenesis) into aspectrum of aggregate structures is thought to cause degenerativediseases referred to as amyloid diseases. TTR undergoes conformationalchanges in order to become amyloidogenic. Partial unfolding exposesstretches of largely uncharged hydrophobic residues in an extendedconformation that efficiently misassemble into largely unstructuredspherical aggregates that ultimately undergo conformation conversioninto cross-beta sheet amyloid structures.

Unless otherwise apparent from context, reference to transthyretin (TTR)or its fragments or domains includes the natural human amino acidsequences including isoforms, mutants, and allelic variants thereof.Exemplary TTR polypeptide sequences are designated by Accession NumbersP02766.1 (UniProt) (SEQ ID NO:38), AAB35639.1 (GenBank) (SEQ ID NO:39),AAB35640.1 (GenBank) (SEQ ID NO:40), and ABI63351.1 (GenBank) (SEQ IDNO:41). Residues are numbered according to Swiss Prot P02766.1, with thefirst amino acid of the mature protein (i.e., not including the 20 aminoacid signal sequence) designated residue 1. In any other TTR protein,residues are numbered according to the corresponding residues inP02766.1 on maximum alignment.

III. Transthyretin Amyloidosis

Transthyretin (TTR) amyloidosis is a systemic disorder characterized bypathogenic, misfolded TTR and the extracellular deposition of amyloidfibrils composed of TTR. TTR amyloidosis is generally caused bydestabilization of the native TTR tetramer form (due to environmental orgenetic conditions), leading to dissociation, misfolding, andaggregation of TTR into amyloid fibrils that accumulate in variousorgans and tissues, causing progressive dysfunction. See, e.g., Almeidaand Saraiva, FEBS Letters 586:2891-2896 (2012); Ando et al., OrphanetJournal of Rare Diseases 8:31 (2013).

In humans, both wild-type TTR tetramers and mixed tetramers comprised ofmutant and wild-type subunits can dissociate, misfold, and aggregate,with the process of amyloidogenesis leading to the degeneration ofpost-mitotic tissue. Thus, TTR amyloidoses encompass diseases caused bypathogenic misfolded TTR resulting from mutations in TTR or resultingfrom non-mutated, misfolded TTR.

For example, senile systemic amyloidosis (SSA) and senile cardiacamyloidosis (SCA) are age-related types of amyloidosis that result fromthe deposition of wild-type TTR amyloid outside and within thecardiomyocytes of the heart. TTR amyloidosis is also the most commonform of hereditary (familial) amyloidosis, which is caused by mutationsthat destabilize the TTR protein. The TTR amyloidoses associated withpoint mutations in the TTR gene include familial amyloid polyneuropathy(FAP), familial amyloid cardiomyopathy (FAC), and the rare centralnervous system selective amyloidosis (CNSA). Patients with hereditary(familial) TTR amyloidosis are almost always heterozygotes, meaning thatthe TTR tetramers are composed of mutant and/or wild-type TTR subunits,generally statistically distributed. Hereditary (familial) versions ofTTR amyloidosis are generally autosomal dominant and are typicallyearlier onset than the sporadic diseases (SSA and SCA).

There are over 100 mutations in the gene encoding TTR that have beenimplicated in the autosomal dominant disorders FAP and FAC. See, e.g.,US 2014/0056904; Saraiva, Hum. Mutat. 17(6):493-503 (2001); Damas andSaraiva, J. Struct. Biol. 130:290-299; Dwulet and Benson, Biochem.Biophys. Res. Commun. 114:657-662 (1983). These amyloid-causingmutations are distributed throughout the entire molecule of TTR.Generally, the more destabilizing the mutant subunits are to the TTRtetramer structure, the earlier the onset of amyloid disease. Thepathogenic potential of a TTR variant is generally determined by acombination of its instability and its cellular secretion efficiency.The initial pathology caused by some TTR variants comes from theirselective destruction of cardiac tissue, whereas that from other TTRvariants comes from compromising the peripheral and autonomic nervoussystem. The tissue damage caused by TTR amyloidogenesis appear to stemlargely from the toxicity of small, diffusible TTR aggregates, althoughaccumulation of extracellular amyloid may contribute and almostcertainly compromises organ structure in the late stages of the TTRamyloidosis.

TTR amyloidosis presents in many different forms, with considerablephenotypic variation across individuals and geographic locations. Forexample, TTR amyloidosis can present as a progressive, axonal sensoryautonomic and motor neuropathy. TTR amyloidosis can also present as aninfiltrative cardiomyopathy.

The age at onset of disease-related symptoms varies between the secondand ninth decades of life, with great variations across differentpopulations. The multisystem involvement of TTR amyloidosis is a clue toits diagnosis. For example, TTR amyloidosis diagnosis is considered whenone or several of the following are present: (1) family history ofneuropathic disease, especially associated with heart failure; (2)neuropathic pain or progressive sensory disturbances of unknownetiology; (3) carpal tunnel syndrome without obvious cause, particularlyif it is bilateral and requires surgical release; (4) gastrointestinalmotility disturbances or autonomic nerve dysfunction of unknown etiology(e.g., erectile dysfunction, orthostatic hypotension, neurogenicgladder); (5) cardiac disease characterized by thickened ventricularwalls in the absence of hypertension; (6) advanced atrio-ventricularblock of unknown origin, particularly when accompanied by a thickenedheart; and (6) vitreous body inclusions of the cotton-wool type. SeeAndo et al., Orphanet Journal of Rare Diseases 8:31 (2013). Othersymptoms can include, for example, polyneuropathy, sensory loss, pain,weakness in lower limbs, dyshidrosis, diarrhea, constipation, weightloss, and urinary incontinence/retention.

Diagnosis of TTR amyloidosis typically relies on target organ biopsies,followed by histological staining of the excised tissue with theamyloid-specific dye, Congo red. If a positive test for amyloid isobserved, immunohistochemical staining for TTR is subsequently performedto ensure that the precursor protein responsible for amyloid formationis indeed TTR. For familial forms of the diseases, demonstration of amutation in the gene encoding TTR is then needed before diagnosis can bemade. This can be accomplished, for example, through isoelectricfocusing electrophoresis, polymerase chain reaction, or laserdissection/liquid chromatography-tandem mass spectrometry. See, e.g., US2014/0056904; Ruberg and Berk, Circulation 126:1286-1300 (2012); Ando etal., Orphanet Journal of Rare Diseases 8:31 (2013).

IV. Antibodies

A. Binding Specificity and Functional Properties

The invention provides monoclonal antibodies binding to transthyretin(TTR) protein, more specifically, to epitopes within amino acid residues89-97 (SEQ ID NO:42) of TTR. Such epitopes are buried in the native TTRtetramer and exposed in monomeric, misfolded, aggregated, or fibrilforms of TTR.

An antibody designated 6C1 is such an exemplary mouse antibody. Thisantibody specifically binds within amino acid residues 89-97 (SEQ IDNO:42) of TTR. This antibody is further characterized by its ability tobind to monomeric, misfolded, aggregated, or fibril forms of TTR but notto native tetrameric forms of TTR. In addition, this antibody ischaracterized by its immunoreactivity on TTR-mediated amyloidosiscardiac tissue but not on healthy cardiac tissue. Ability to bind tospecific proteins or fragments thereof may be demonstrated usingexemplary assay formats provided in the examples.

Some antibodies bind to the same or overlapping epitope as an antibodydesignated 6C1. The sequences of the heavy and light chain maturevariable regions of 6C1 are designated SEQ ID NOS: 1 and 13,respectively. Other antibodies having such a binding specificity can beproduced by immunizing mice with TTR, or a portion thereof including thedesired epitope (e.g., SEQ ID NO:42), and screening resulting antibodiesfor binding to monomeric TTR or a peptide comprising SEQ ID NO:42,optionally in competition with an antibody having the variable regionsof mouse 6C1 (IgG1,kappa). Fragments of TTR including the desiredepitope can be linked to a carrier that helps elicit an antibodyresponse to the fragment and/or be combined with an adjuvant that helpselicit such a response. Such antibodies can be screened for differentialbinding to wild-type, monomeric versions of TTR or a fragment thereof(e.g., SEQ ID NO:38) compared with mutants of specified residues.Screening against such mutants more precisely defines the bindingspecificity to allow identification of antibodies whose binding isinhibited by mutagenesis of particular residues and which are likely toshare the functional properties of other exemplified antibodies. Themutations can be systematic replacement substitution with alanine (orserine if an alanine is present already) one residue at a time, or morebroadly spaced intervals, throughout the target or throughout a sectionthereof in which an epitope is known to reside. If the same set ofmutations significantly reduces the binding of two antibodies, the twoantibodies bind the same epitope.

Antibodies having the binding specificity of a selected murine antibody(e.g., 6C1) can also be produced using a variant of the phage displaymethod. See Winter, WO 92/20791. This method is particularly suitablefor producing human antibodies. In this method, either the heavy orlight chain variable region of the selected murine antibody is used as astarting material. If, for example, a light chain variable region isselected as the starting material, a phage library is constructed inwhich members display the same light chain variable region (i.e., themurine starting material) and a different heavy chain variable region.The heavy chain variable regions can for example be obtained from alibrary of rearranged human heavy chain variable regions. A phageshowing strong specific binding (e.g., at least 10⁸ and preferably atleast 10⁹M⁻¹) for monomeric TTR or a fragment thereof (e.g., amino acidresidues 89-97) is selected. The heavy chain variable region from thisphage then serves as a starting material for constructing a furtherphage library. In this library, each phage displays the same heavy chainvariable region (i.e., the region identified from the first displaylibrary) and a different light chain variable region. The light chainvariable regions can be obtained for example from a library ofrearranged human variable light chain regions. Again, phage showingstrong specific binding for monomeric TTR or a fragment thereof (e.g.,amino acid residues 89-97) are selected. The resulting antibodiesusually have the same or similar epitope specificity as the murinestarting material.

Other antibodies can be obtained by mutagenesis of cDNA encoding theheavy and light chains of an exemplary antibody, such as 6C1. Monoclonalantibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to 6C1 in amino acid sequence of the mature heavy and/or lightchain variable regions and maintain its functional properties, and/orwhich differ from the respective antibody by a small number offunctionally inconsequential amino acid substitutions (e.g.,conservative substitutions), deletions, or insertions are also includedin the invention. Monoclonal antibodies having at least one or all sixCDR(s) as defined by conventional definition, but preferably Kabat, thatare 90%, 95%, 99% or 100% identical to corresponding CDRs of 6C1 arealso included.

The invention also provides antibodies having some or all (e.g., 3, 4,5, and 6) CDRs entirely or substantially from 6C1. Such antibodies caninclude a heavy chain variable region that has at least two, and usuallyall three, CDRs entirely or substantially from the heavy chain variableregion of 6C1 and/or a light chain variable region having at least two,and usually all three, CDRs entirely or substantially from the lightchain variable region of 6C1. The antibodies can include both heavy andlight chains. A CDR is substantially from a corresponding 6C1 CDR whenit contains no more than 4, 3, 2, or 1 substitutions, insertions, ordeletions, except that CDR-H2 (when defined by Kabat) can have no morethan 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Suchantibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identity to 6C1 in the amino acid sequence of the mature heavy and/orlight chain variable regions and maintain their functional properties,and/or differ from 6C1 by a small number of functionally inconsequentialamino acid substitutions (e.g., conservative substitutions), deletions,or insertions.

Some antibodies identified by such assays can bind to monomeric,misfolded, aggregated, or fibril forms of TTR but not to nativetetrameric forms of TTR, as described in the examples or otherwise.Likewise, some antibodies are immunoreactive on TTR-mediated amyloidosistissue but not on healthy tissue.

Some antibodies can inhibit or reduce aggregation of TTR, inhibit orreduce TTR fibril formation, reduce or clear TTR deposits or aggregatedTTR, or stabilize non-toxic conformations of TTR in an animal model orclinical trial. Some antibodies can treat, effect prophylaxis of, ordelay the onset of a TTR amyloidosis as shown in an animal model orclinical trial. Exemplary animal models for testing activity against aTTR amyloidosis include those described in Kohno et al., Am. J. Path.150(4):1497-1508 (1997); Teng et al., Laboratory Investigations81:385-396 (2001); Wakasugi et al., Proc. Japan Acad. 63B:344-347(1987); Shimada et al., Mol. Biol. Med. 6:333-343 (1989); Nagata et al.,J Biochem. 117:169-175 (1995); Sousa et al., Am. J. Path. 161:1935-1948(2002); and Santos et al., Neurobiology of Aging 31:280-289 (2010).

B. Non-Human Antibodies

The production of other non-human antibodies, e.g., murine, guinea pig,primate, rabbit or rat, against monomeric TTR or a fragment thereof(e.g., amino acid residues 89-97) can be accomplished by, for example,immunizing the animal with TTR or a fragment thereof. See Harlow & Lane,Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated byreference for all purposes). Such an immunogen can be obtained from anatural source, by peptide synthesis, or by recombinant expression.Optionally, the immunogen can be administered fused or otherwisecomplexed with a carrier protein. Optionally, the immunogen can beadministered with an adjuvant. Several types of adjuvant can be used asdescribed below. Complete Freund's adjuvant followed by incompleteadjuvant is preferred for immunization of laboratory animals. Rabbits orguinea pigs are typically used for making polyclonal antibodies. Miceare typically used for making monoclonal antibodies. Antibodies arescreened for specific binding to monomeric TTR or an epitope within TTR(e.g., an epitope comprising one or more of amino acid residues 89-97).Such screening can be accomplished by determining binding of an antibodyto a collection of monomeric TTR variants, such as TTR variantscontaining amino acid residues 89-97 or mutations within these residues,and determining which TTR variants bind to the antibody. Binding can beassessed, for example, by Western blot, FACS or ELISA.

C. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which CDRsfrom a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No.6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. Thus, a humanized antibody is an antibody having at leastthree, four, five or all CDRs entirely or substantially from a donorantibody and variable region framework sequences and constant regions,if present, entirely or substantially from human antibody sequences.Similarly a humanized heavy chain has at least one, two and usually allthree CDRs entirely or substantially from a donor antibody heavy chain,and a heavy chain variable region framework sequence and heavy chainconstant region, if present, substantially from human heavy chainvariable region framework and constant region sequences. Similarly ahumanized light chain has at least one, two and usually all three CDRsentirely or substantially from a donor antibody light chain, and a lightchain variable region framework sequence and light chain constantregion, if present, substantially from human light chain variable regionframework and constant region sequences. Other than nanobodies and dAbs,a humanized antibody comprises a humanized heavy chain and a humanizedlight chain. A CDR in a humanized antibody is substantially from acorresponding CDR in a non-human antibody when at least 85%, 90%, 95% or100% of corresponding residues (as defined by any conventionaldefinition but preferably defined by Kabat) are identical between therespective CDRs. The variable region framework sequences of an antibodychain or the constant region of an antibody chain are substantially froma human variable region framework sequence or human constant regionrespectively when at least 85%, 90%, 95% or 100% of correspondingresidues defined by any conventional definition but preferably definedby Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a mouse antibody, they can also be made withless than all CDRs (e.g., at least 3, 4, or 5 CDRs) from a mouseantibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos etal., J. of Mol. Biol., 320: 415-428, 2002; Iwahashi et al., Mol.Immunol. 36:1079-1091, 1999; Tamura et al, J. Immunol., 164:1432-1441,2000).

In some antibodies only part of the CDRs, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting antigen andnot in the SDRs can be identified based on previous studies (for exampleresidues H60-H65 in CDR H2 are often not required), from regions ofKabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol.Biol. 196:901, 1987), by molecular modeling and/or empirically, or asdescribed in Gonzales et al., Mol. Immunol. 41: 863, 2004. In suchhumanized antibodies at positions in which one or more donor CDRresidues is absent or in which an entire donor CDR is omitted, the aminoacid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity. However, substitutionscan also cause changes of affinity, and significant reductions inaffinity are preferably avoided. Positions for substitution within CDRsand amino acids to substitute can also be selected empirically.

The human acceptor antibody sequences can optionally be selected fromamong the many known human antibody sequences to provide a high degreeof sequence identity (e.g., 65-85% identity) between a human acceptorsequence variable region frameworks and corresponding variable regionframeworks of a donor antibody chain.

An example of an acceptor sequence for the heavy chain is the humanmature heavy chain variable region with NCBI accession code ADX65650(SEQ ID NO:3). This acceptor sequence includes two CDRs having the samecanonical form as mouse 6C1 heavy chain. An examples of an acceptorsequence for the light chain is the human mature light chain variableregion with NCBI accession code ABI74084 (SEQ ID NO:15). This acceptorsequence includes two CDRs having the same canonical form as mouse 6C1light chain.

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly;    -   (2) is adjacent to a CDR region or within a CDR as defined by        Chothia but not Kabat;    -   (3) otherwise interacts with a CDR region (e.g., is within about        6 Å of a CDR region), (e.g., identified by modeling the light or        heavy chain on the solved structure of a homologous known        immunoglobulin chain); or    -   (4) is a residue participating in the VL-VH interface.

Framework residues from classes (1) through (3) as defined by Queen,U.S. Pat. No. 5,530,101, are sometimes alternately referred to ascanonical and vernier residues. Framework residues that help define theconformation of a CDR loop are sometimes referred to as canonicalresidues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton &Martin, J. Mol. Biol. 263:800-815 (1996)). Framework residues thatsupport antigen-binding loop conformations and play a role infine-tuning the fit of an antibody to antigen are sometimes referred toas vernier residues (Foote & Winter, J. Mol. Biol 224:487-499 (1992)).

Other framework residues that are candidates for substitution areresidues creating a potential glycosylation site. Still other candidatesfor substitution are acceptor human framework amino acids that areunusual for a human immunoglobulin at that position. These amino acidscan be substituted with amino acids from the equivalent position of themouse donor antibody or from the equivalent positions of more typicalhuman immunoglobulins.

Exemplary humanized antibodies are humanized forms of the mouse 6C1antibody, designated Hu6C1. The mouse antibody comprises mature heavyand light chain variable regions having amino acid sequences comprisingSEQ ID NO:1 and SEQ ID NO:13, respectively. The invention provides sixexemplified humanized mature heavy chain variable regions: Hu6C1VHv1(SEQ ID NO:4), Hu6C1VHv1b (SEQ ID NO:5), Hu6C1VHv2 (SEQ ID NO:6),Hu6C1VHv2b (SEQ ID NO:7), Hu6C1VHv3 (SEQ ID NO:8), and Hu6C1VHv3b (SEQID NO:9). The invention further provides two exemplified human maturelight chain variable regions: Hu6C1VLv1 (SEQ ID NO:16) and Hu6C1VLv2(SEQ ID NO:17). FIGS. 1 and 2 show alignments of the heavy chainvariable region and light chain variable region, respectively, of 6C1,mouse model antibodies, human acceptor antibodies, and humanizedantibody versions of 6C1.

For reasons such as possible influence on CDR conformation and/orbinding to antigen, mediating interaction between heavy and lightchains, interaction with the constant region, being a site for desiredor undesired post-translational modification, being an unusual residuefor its position in a human variable region sequence and thereforepotentially immunogenic, getting aggregation potential, and otherreasons, the following ten variable region framework positions wereconsidered as candidates for substitutions in the six exemplified humanmature light chain variable regions and the two exemplified human matureheavy chain variable regions, as further specified in the examples: L2,L45, H19, H44, H49, H76, H77, H82(a), H83, and H89.

Here, as elsewhere, the first-mentioned residue is the residue of ahumanized antibody formed by grafting Kabat CDRs or a composite ChothiaKabat CDR in the case of CDR-H1 into a human acceptor framework, and thesecond-mentioned residue is a residue being considered for replacingsuch residue. Thus, within variable region frameworks, the firstmentioned residue is human, and within CDRs, the first mentioned residueis mouse.

Exemplified antibodies include any permutations or combinations of theexemplified mature heavy and light chain variable regions (e.g.,Hu6C1VHv1/VLv1 or H1L1, Hu6C1VHv1b/VLv1 or H1bL1, Hu6C1VHv1/VLv2 orH1L2, Hu6C1VHv1b/VLv2 or H1bL2, Hu6C1VHv2/VLv1 or H2L1, Hu6C1VHv2b/VLv1or H2bL1, Hu6C1VHv2/VLv2 or H2L2, Hu6C1VHv2b/VLv2 or H2bL2,Hu6C1VHv3/VLv1 or H3L1, Hu6C1VHv3b/VLv1 or H3bL1, Hu6C1VHv3/VLv2 orH3L2, and Hu6C1VHv3b/VLv2 or H3bL2).

The invention provides variants of humanized antibodies in which thehumanized mature heavy chain variable region shows at least 90%, 95%,96%, 97%, 98%, or 99% identity to SEQ ID NOs:4-9, and the humanizedmature light chain variable region shows at least 90%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NOs:16 or 17. In some such antibodies atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of the backmutations or othermutations in SEQ ID NOs:4-9, 16, and 17 are retained.

In some antibodies, at least one of positions H19, H44, H49, H76, H77,H82(a), H83, and H89 in the V_(H) region is occupied by K, R, A, S, T,S, K, and V, respectively. In some antibodies, position H77 in the V_(H)region is occupied by T, as in Hu6C1VHv1. In some antibodies, positionsH49 and H77 in the V_(H) region are occupied by A and T, respectively,as in Hu6C1VHv1b. In some antibodies, positions H76, H77, and H82(a) inthe V_(H) region are occupied by S, T, and S, respectively, as inHu6C1VHv2. In some antibodies, positions H49, H76, H77, and H82(a) inthe V_(H) region are occupied by A, S, T, and S, respectively, as inHu6C1VHv2b. In some antibodies, positions H19, H44, H77, H83, and H89 inthe V_(H) region are occupied by K, R, T, K, and M, respectively, as inHu6C1VHv3. In some antibodies, positions H19, H44, H49, H77, H83, andH89 in the V_(H) region are occupied by K, R, A, T, K, and M,respectively, as in Hu6C1VHv3b. In some antibodies, position L45 in theVL region is occupied by K. In some antibodies, position L2 in the V_(L)region is occupied by I. In some antibodies, one or both of positions L2and L45 in the V_(L) region are occupied by V and K, respectively, as inHu6C1VLv1. In some antibodies, one or both of positions L2 and L45 inthe V_(L) region are occupied by I and K, respectively, as in Hu6C1VLv2.The CDR regions of such humanized antibodies can be identical orsubstantially identical to the CDR regions of the 6C1 mouse donorantibody. The CDR regions can be defined by any conventional definition(e.g., Chothia, or composite of Chothia and Kabat) but are preferably asdefined by Kabat.

Variable regions framework positions are in accordance with Kabatnumbering unless otherwise stated. Other such variants typically differfrom the sequences of the exemplified Hu6C1 antibodies by a small number(e.g., typically no more than 1, 2, 3, 5, 10, or 15) of replacements,deletions or insertions. Such differences are usually in the frameworkbut can also occur in the CDRs.

A possibility for additional variation in humanized 6C1 variants isadditional backmutations in the variable region frameworks. Many of theframework residues not in contact with the CDRs in the humanized mAb canaccommodate substitutions of amino acids from the correspondingpositions of the donor mouse mAb or other mouse or human antibodies, andeven many potential CDR-contact residues are also amenable tosubstitution. Even amino acids within the CDRs may be altered, forexample, with residues found at the corresponding position of the humanacceptor sequence used to supply variable region frameworks. Inaddition, alternate human acceptor sequences can be used, for example,for the heavy and/or light chain. If different acceptor sequences areused, one or more of the backmutations recommended above may not beperformed because the corresponding donor and acceptor residues arealready the same without backmutations.

Preferably, replacements or backmutations in Hu6C1 variants (whether ornot conservative) have no substantial effect on the binding affinity orpotency of the humanized mAb, that is, its ability to bind to monomericTTR (e.g., the potency in some or all of the assays described in thepresent examples of the variant humanized 6C1 antibody is essentiallythe same, i.e., within experimental error, as that of murine 6C1).

D. Chimeric and Veneered Antibodies

The invention further provides chimeric and veneered forms of non-humanantibodies, particularly the 6C1 antibodies of the examples.

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions. Veneered forms of the 6C1 antibody are included in theinvention.

E. Human Antibodies

Human antibodies against monomeric TTR or a fragment thereof (e.g.,amino acid residues 89-97 (SEQ ID NO:42) of TTR) are provided by avariety of techniques described below. Some human antibodies areselected by competitive binding experiments, by the phage display methodof Winter, above, or otherwise, to have the same epitope specificity asa particular mouse antibody, such as one of the mouse monoclonalantibodies described in the examples. Human antibodies can also bescreened for particular epitope specificity by using only a fragment ofTTR, such as a TTR variant containing only amino acid residues 89-97 ofTTR, as the target antigen, and/or by screening antibodies against acollection of TTR variants, such as TTR variants containing variousmutations within amino acid residues 89-97 of TTR.

Methods for producing human antibodies include the trioma method ofOestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No.4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, use oftransgenic mice including human immunoglobulin genes (see, e.g., Lonberget al., WO93/12227 (1993); U.S. Pat. No. 5,877,397; U.S. Pat. No.5,874,299; U.S. Pat. No. 5,814,318; U.S. Pat. No. 5,789,650; U.S. Pat.No. 5,770,429; U.S. Pat. No. 5,661,016; U.S. Pat. No. 5,633,425; U.S.Pat. No. 5,625,126; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,545,806;Neuberger, Nat. Biotechnol. 14:826 (1996); and Kucherlapati, WO 91/10741(1991)) and phage display methods (see, e.g., Dower et al., WO 91/17271;McCafferty et al., WO 92/01047; U.S. Pat. No. 5,877,218; U.S. Pat. No.5,871,907; U.S. Pat. No. 5,858,657; U.S. Pat. No. 5,837,242; U.S. Pat.No. 5,733,743; and U.S. Pat. No. 5,565,332).

F. Selection of Constant Region

The heavy and light chain variable regions of chimeric, veneered orhumanized antibodies can be linked to at least a portion of a humanconstant region. The choice of constant region depends, in part, whetherantibody-dependent cell-mediated cytotoxicity, antibody dependentcellular phagocytosis and/or complement dependent cytotoxicity aredesired. For example, human isotopes IgG1 and IgG3 havecomplement-dependent cytotoxicity and human isotypes IgG2 and IgG4 donot. Human IgG1 and IgG3 also induce stronger cell mediated effectorfunctions than human IgG2 and IgG4. Light chain constant regions can belambda or kappa.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering is used in this paragraphfor the constant region) for increasing the half-life of an antibody.Substitution at any or all of positions 234, 235, 236 and/or 237 reduceaffinity for Fcγ receptors, particularly FcγRT receptor (see, e.g., U.S.Pat. No. 6,624,821). An alanine substitution at positions 234, 235, and237 of human IgG1 can be used for reducing effector functions. Someantibodies have alanine substitution at positions 234, 235 and 237 ofhuman IgG1 for reducing effector functions. Optionally, positions 234,236 and/or 237 in human IgG2 are substituted with alanine and position235 with glutamine (see, e.g., U.S. Pat. No. 5,624,821). In someantibodies, a mutation at one or more of positions 241, 264, 265, 270,296, 297, 322, 329, and 331 by EU numbering of human IgG1 is used. Insome antibodies, a mutation at one or more of positions 318, 320, and322 by EU numbering of human IgG1 is used. In some antibodies, positions234 and/or 235 are substituted with alanine and/or position 329 issubstituted with glycine. In some antibodies, positions 234 and 235 aresubstituted with alanine, such as in SEQ ID NO:27. In some antibodies,the isotype is human IgG2 or IgG4.

An exemplary human light chain kappa constant region has the amino acidsequence of SEQ ID NO:28. The N-terminal arginine of SEQ ID NO:28 can beomitted, in which case light chain kappa constant region has the aminoacid sequence of SEQ ID NO:29. An exemplary human IgG1 heavy chainconstant region has the amino acid sequence of SEQ ID NO:25 (with orwithout the C-terminal lysine). Antibodies can be expressed as tetramerscontaining two light and two heavy chains, as separate heavy chains,light chains, as Fab, Fab′, F(ab′)2, and Fv, or as single chainantibodies in which heavy and light chain mature variable domains arelinked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype bind to a non-polymorphic region of a one or more otherisotypes. Thus, for example, another heavy chain constant region is ofIgG1 G1m3 allotype and has the amino acid sequence of SEQ ID NO:26.Another heavy chain constant region of the IgG1 G1m3 allotype has theamino acid sequence of SEQ ID NO:27 (with or without the C-terminallysine). Reference to a human constant region includes a constant regionwith any natural allotype or any permutation of residues occupyingpositions in natural allotypes.

G. Expression of Recombinant Antibodies

A number of methods are known for producing chimeric and humanizedantibodies using an antibody-expressing cell line (e.g., hybridoma). Forexample, the immunoglobulin variable regions of antibodies can be clonedand sequenced using well known methods. In one method, the heavy chainvariable VH region is cloned by RT-PCR using mRNA prepared fromhybridoma cells. Consensus primers are employed to the VH region leaderpeptide encompassing the translation initiation codon as the 5′ primerand a g2b constant regions specific 3′ primer. Exemplary primers aredescribed in U.S. patent publication US 2005/0009150 by Schenk et al.(hereinafter “Schenk”). The sequences from multiple, independentlyderived clones can be compared to ensure no changes are introducedduring amplification. The sequence of the VH region can also bedetermined or confirmed by sequencing a VH fragment obtained by 5′ RACERT-PCR methodology and the 3′ g2b specific primer.

The light chain variable VL region can be cloned in an analogous manner.In one approach, a consensus primer set is designed for amplification ofVL regions using a 5′ primer designed to hybridize to the VL regionencompassing the translation initiation codon and a 3′ primer specificfor the Ck region downstream of the V-J joining region. In a secondapproach, 5′RACE RT-PCR methodology is employed to clone a VL encodingcDNA. Exemplary primers are described in Schenk, supra. The clonedsequences are then combined with sequences encoding human (or othernon-human species) constant regions. Exemplary sequences encoding humanconstant regions include SEQ ID NO:46, which encodes a human IgG1constant region, and SEQ ID NOs:47 and 48, which encode a human kappalight chain constant region.

In one approach, the heavy and light chain variable regions arere-engineered to encode splice donor sequences downstream of therespective VDJ or VJ junctions and are cloned into a mammalianexpression vector, such as pCMV-hγ1 for the heavy chain and pCMV-Mcl forthe light chain. These vectors encode human γ1 and Ck constant regionsas exonic fragments downstream of the inserted variable region cassette.Following sequence verification, the heavy chain and light chainexpression vectors can be co-transfected into CHO cells to producechimeric antibodies. Conditioned media is collected 48 hourspost-transfection and assayed by western blot analysis for antibodyproduction or ELISA for antigen binding. The chimeric antibodies arehumanized as described above.

Chimeric, veneered, humanized, and human antibodies are typicallyproduced by recombinant expression. Recombinant polynucleotideconstructs typically include an expression control sequence operablylinked to the coding sequences of antibody chains, including naturallyassociated or heterologous expression control elements, such as apromoter. The expression control sequences can be promoter systems invectors capable of transforming or transfecting eukaryotic orprokaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences and the collection andpurification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g., ampicillinresistance or hygromycin resistance, to permit detection of those cellstransformed with the desired DNA sequences.

E. coli is one prokaryotic host useful for expressing antibodies,particularly antibody fragments. Microbes, such as yeast, are alsouseful for expression. Saccharomyces is a yeast host with suitablevectors having expression control sequences, an origin of replication,termination sequences, and the like as desired. Typical promotersinclude 3-phosphoglycerate kinase and other glycolytic enzymes.Inducible yeast promoters include, among others, promoters from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

Mammalian cells can be used for expressing nucleotide segments encodingimmunoglobulins or fragments thereof. See Winnacker, From Genes toClones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped, and include CHO cell lines, various COS cell lines, HeLacells, HEK293 cells, L cells, and non-antibody-producing myelomasincluding Sp2/0 and NS0. The cells can be nonhuman. Expression vectorsfor these cells can include expression control sequences, such as anorigin of replication, a promoter, an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Expression control sequences caninclude promoters derived from endogenous genes, cytomegalovirus, SV40,adenovirus, bovine papillomavirus, and the like. See Co et al., J.Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No.5,849,992). Suitable transgenes include coding sequences for lightand/or heavy chains operably linked with a promoter and enhancer from amammary gland specific gene, such as casein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by methods depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics, or viral-based transfection can be used forother cellular hosts. Other methods used to transform mammalian cellsinclude the use of polybrene, protoplast fusion, liposomes,electroporation, and microinjection. For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected of FACS-based single-cell cloning to generatemonoclonal lines. Specific productivities above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, can be used. Antibodies produced by single cell clones can alsobe tested for turbidity, filtration properties, PAGE, IEF, UV scan,HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, andbinding assay, such as ELISA or Biacore. A selected clone can then bebanked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, HPLC purification,column chromatography, gel electrophoresis and the like (see generally,Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies can be employed,including codon optimization, selection of promoters, selection oftranscription elements, selection of terminators, serum-free single cellcloning, cell banking, use of selection markers for amplification ofcopy number, CHO terminator, or improvement of protein titers (see,e.g., U.S. Pat. No. 5,786,464; U.S. Pat. No. 6,114,148; U.S. Pat. No.6,063,598; U.S. Pat. No. 7,569,339; WO2004/050884; WO2008/012142;WO2008/012142; WO2005/019442; WO2008/107388; WO2009/027471; and U.S.Pat. No. 5,888,809).

H. Antibody Screening Assays

Antibodies can be subject to several screens including binding assays,functional screens, screens in animal models of diseases associated withTTR deposits, and clinical trials. Binding assays test for specificbinding and, optionally, affinity and epitope specificity to monomericTTR or a fragment thereof. For example, binding assays can screen forantibodies that bind to amino acid residues 89-97 (SEQ ID NO:42) of TTR,which is an epitope that is buried in the native TTR tetramer andexposed in monomeric, misfolded, aggregated, or fibril forms of TTR.Antibodies can also be screened for the ability to bind pre-fibrillar,non-native conformations of TTR and TTR amyloid fibrils but not nativeTTR conformations. For example, antibodies can be screened for theability to bind to monomeric forms of TTR created by dissociation ordisaggregation of native tetrameric TTR, and can be counter-screenedagainst native tetrameric TTR, as described in the examples orotherwise. Likewise, antibodies can also be screened for theirimmunoreactivity on TTR-mediated amyloidosis tissue but not on healthytissue. Such screens are sometimes performed in competition with anexemplary antibody, such as an antibody having the variable regions of6C1 or IgG1 kappa isotype. Optionally, either the antibody or TTR targetis immobilized in such assay.

Functional assays can be performed in cellular models including cellsnaturally expressing TTR or transfected with DNA encoding TTR or afragment thereof. Suitable cells include cells derived from cardiactissue or other tissues affected by TTR amyloidogenesis. Cells can bescreened for reduced levels of monomeric, misfolded, aggregated, orfibril forms of TTR (e.g., by Western blotting or immunoprecipitation ofcell extracts or supernatants) or reduced toxicity attributable tomonomeric, misfolded, aggregated, or fibril forms of TTR. For example,antibodies can tested for the ability to inhibit or reduce aggregationof TTR, inhibit or reduce TTR fibril formation, reduce TTR deposits,clear aggregated TTR, or stabilize non-toxic conformations of TTR.

Other functional assays can be performed in solution, such as testingwhether an antibody is capable of disrupting or reducing TTR fibrilformation when monomeric TTR or misfolded TTR intermediates in solutionare contacted with the antibody. The extent of fibril formation can beprobed by turbidity measurements, for example, at 400 nm on a UV-visiblespectrometer equipped with a temperature control unit. Thioflavin-T canalso be used to assess the extent of amyloid fibril formation. Forexample, a five-fold molar excess of Thioflavin-T can be added to TTRsamples and left at room temperature for 30 minutes before measurementsare taken. Thioflavin-T fluorescence can be monitored using aspectrofluorimeter. See US 2014/0056904.

Animal model screens test the ability of the antibody to therapeuticallyor prophylactically treat signs or symptoms in an animal modelsimulating a human disease associated with accumulation of TTR or TTRdeposits. Such diseases include types of TTR amyloidosis, such as senilesystemic amyloidosis (SSA), senile cardiac amyloidosis (SCA), familialamyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), andcentral nervous system selective amyloidosis (CNSA). Suitable signs orsymptoms that can be monitored include the presence and extent ofamyloid deposits in various tissues, such as the gastrointestinal tractor heart. The extent of reduction of amyloid deposits can be determinedby comparison with an appropriate control, such the level of TTR amyloiddeposits in control animals that have received a control antibody (e.g.,an isotype matched control antibody), a placebo, or no treatment at all.An exemplary animal model for testing activity against a TTR amyloidosisis a mouse model carrying a null mutation at the endogenous mouse Ttrlocus and the human mutant TTR gene comprising a V30M mutation that isassociated with familial amyloidotic polyneuropathy. See, e.g., Kohno etal., Am. J. Path. 150(4):1497-1508 (1997); Cardoso and Saraiva, FASEB20(2):234-239 (2006). Similar models also exist, including other modelsfor familial versions of TTR amyloidosis and models for sporadicversions of TTR amyloidosis. See, e.g., Teng et al., Lab. Invest. 81(3):385-396 (2001); Ito and Maeda, Mouse Models of TransthyretinAmyloidosis, in Recent Advances in Transthyretin Evolution, Structure,and Biological Functions, pp. 261-280 (2009) (Springer BerlinHeidelberg). Transgenic animals can include a human TTR transgene, suchas a TTR transgene with a mutation associated with TTR amyloidosis or awild-type TTR transgene. To facilitate testing in animal models,chimeric antibodies having a constant region appropriate for the animalmodel can be used (e.g., mouse-rat chimeras could be used for testingantibodies in rats). It can be concluded that a humanized version of anantibody will be effective if the corresponding mouse antibody orchimeric antibody is effective in an appropriate animal model and thehumanized antibody has similar binding affinity (e.g., withinexperimental error, such as by a factor of 1.5, 2, or 3).

Clinical trials test for safety and efficacy in a human having a diseaseassociated with TTR amyloidosis.

I. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above (e.g., SEQ ID NOS:4-9, 16, and 17).Optionally, such nucleic acids further encode a signal peptide and canbe expressed with the signal peptide linked to the constant region(e.g., signal peptides having amino acid sequences of SEQ ID NOS:49(heavy chain) and 51 (light chain) that can be encoded by SEQ ID NOS:50,respectively (heavy chain) and 52, respectively (light chain)). Codingsequences of nucleic acids can be operably linked with regulatorysequences to ensure expression of the coding sequences, such as apromoter, enhancer, ribosome binding site, transcription terminationsignal, and the like. The nucleic acids encoding heavy and light chainscan occur in isolated form or can be cloned into one or more vectors.The nucleic acids can be synthesized by, for example, solid statesynthesis or PCR of overlapping oligonucleotides. Nucleic acids encodingheavy and light chains can be joined as one contiguous nucleic acid,e.g., within an expression vector, or can be separate, e.g., each clonedinto its own expression vector.

J. Conjugated Antibodies

Conjugated antibodies that specifically bind to antigens exposed inpathogenic forms of TTR but not in native tetrameric forms of TTR, suchas amino acid residues 89-97 (SEQ ID NO:42) of TTR, are useful indetecting the presence of monomeric, misfolded, aggregated, or fibrilforms of TTR; monitoring and evaluating the efficacy of therapeuticagents being used to treat patients diagnosed with a TTR amyloidosis;inhibiting or reducing aggregation of TTR; inhibiting or reducing TTRfibril formation; reducing or clearing TTR deposits; stabilizingnon-toxic conformations of TTR; or treating or effecting prophylaxis ofa TTR amyloidosis in a patient. For example, such antibodies can beconjugated with other therapeutic moieties, other proteins, otherantibodies, and/or detectable labels. See WO 03/057838; U.S. Pat. No.8,455,622.

Conjugated therapeutic moieties can be any agent that can be used totreat, combat, ameliorate, prevent, or improve an unwanted condition ordisease in a patient, such as a TTR amyloidosis. Therapeutic moietiescan include, for example, immunomodulators or any biologically activeagents that facilitate or enhance the activity of the antibody. Animmunomodulator can be any agent that stimulates or inhibits thedevelopment or maintenance of an immunologic response. If suchtherapeutic moieties are coupled to an antibody specific for monomeric,misfolded, aggregated, or fibril forms of TTR, such as the antibodiesdescribed herein, the coupled therapeutic moieties will have a specificaffinity for non-native, pathogenic forms of TTR over native tetramericforms of TTR. Consequently, administration of the conjugated antibodiesdirectly targets tissues comprising pathogenic forms of TTR with minimaldamage to surrounding normal, healthy tissue. This can be particularlyuseful for therapeutic moieties that are too toxic to be administered ontheir own. In addition, smaller quantities of the therapeutic moietiescan be used.

Examples of suitable therapeutic moieties include drugs that reducelevels of TTR, stabilize the native tetrameric structure of TTR, inhibitaggregation of TTR, disrupt TTR fibril or amyloid formation, orcounteract cellular toxicity. See, e.g., Almeida and Saraiva, FEBSLetters 586:2891-2896 (2012); Saraiva, FEBS Letters 498:201-203 (2001);Ando et al., Orphanet Journal of Rare Diseases 8:31 (2013); Ruberg andBerk, Circulation 126:1286-1300 (2012); and Johnson et al., J. Mol.Biol. 421(2-3):185-203 (2012). For example, antibodies can be conjugatedto tafamidis, diflunisal, ALN-TTR01, ALNTTR02, ISIS-TTRRx, doxycycline(doxy), tauroursodeoxycholic acid (TUDCA), Doxy-TUDCA, epigallocatechingallate (EGCG), curcumin, or resveratrol (3,5,4′-trihydroxystilbene).Other representative therapeutic moieties include other agents known tobe useful for treatment, management, or amelioration of a TTRamyloidosis or symptoms of a TTR amyloidosis. See, e.g., Ando et al.,Orphanet Journal of Rare Diseases 8:31 (2013) for common clinicalsymptoms of TTR amyloidosis and typical agents used to treat thosesymptoms.

Antibodies can also be coupled with other proteins. For example,antibodies can be coupled with Fynomers. Fynomers are small bindingproteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They canbe stable and soluble, and they can lack cysteine residues and disulfidebonds. Fynomers can be engineered to bind to target molecules with thesame affinity and specificity as antibodies. They are suitable forcreating multi-specific fusion proteins based on antibodies. Forexample, Fynomers can be fused to N-terminal and/or C-terminal ends ofantibodies to create bi- and tri-specific FynomAbs with differentarchitectures. Fynomers can be selected using Fynomer libraries throughscreening technologies using FACS, Biacore, and cell-based assays thatallow efficient selection of Fynomers with optimal properties. Examplesof Fynomers are disclosed in Grabulovski et al., J. Biol. Chem.282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel.20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner etal., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6):1124-1137 (2013);and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

The antibodies disclosed herein can also be coupled or conjugated to oneor more other antibodies (e.g., to form antibody heteroconjugates). Suchother antibodies can bind to different epitopes within TTR or a portionthereof or can bind to a different target antigen.

Antibodies can also be coupled with a detectable label. Such antibodiescan be used, for example, for diagnosing a TTR amyloidosis, formonitoring progression of a TTR amyloidosis, and/or for assessingefficacy of treatment. Such antibodies are particularly useful forperforming such determinations in subjects having or being susceptibleto a TTR amyloidosis, or in appropriate biological samples obtained fromsuch subjects. Representative detectable labels that may be coupled orlinked to a humanized 6C1 antibody include various enzymes, such ashorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such streptavidin/biotin andavidin/biotin; fluorescent materials, such as umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as luminol; bioluminescent materials, suchas luciferase, luciferin, and aequorin; radioactive materials, such asyttrium⁹⁰ (90Y), radiosilver-111, radiosilver-199, Bismuth²¹³, iodine(¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,), carbon (¹⁴C), sulfur (⁵S), tritium (³H),indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), technetium (⁹⁹Tc), thallium(²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo),xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn,⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin; positronemitting metals using various positron emission tomographies;nonradioactive paramagnetic metal ions; and molecules that areradiolabelled or conjugated to specific radioisotopes.

Linkage of radioisotopes to antibodies may be performed withconventional bifunction chelates. For radiosilver-111 andradiosilver-199 linkage, sulfur-based linkers may be used. See Hazra etal., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes mayinvolve reducing the immunoglobulin with ascorbic acid. Forradioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be usedand will react with such isotopes to form 111In-ibritumomab tiuxetan and90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother.Pharmacol., 48 Suppl 1:S91-S95 (2001).

Therapeutic moieties, other proteins, other antibodies, and/ordetectable labels may be coupled or conjugated, directly or indirectlythrough an intermediate (e.g., a linker), to a murine, chimeric,veneered, or humanized 6C1 antibody using techniques known in the art.See e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstromet al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies 84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy,” in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press1985); and Thorpe et al., Immunol. Rev., 62:119-58 (1982). Suitablelinkers include, for example, cleavable and non-cleavable linkers.Different linkers that release the coupled therapeutic moieties,proteins, antibodies, and/or detectable labels under acidic or reducingconditions, on exposure to specific proteases, or under other definedconditions can be employed.

V. Therapeutic Applications

The above antibodies can be used for treating or effecting prophylaxisof a disease in a patient having or at risk for the disease mediated atleast in part by transthyretin (TTR), and particularly by monomeric,misfolded, aggregated, or fibril forms of TTR. Although an understandingof mechanism is not required for practice, it is believed that any orall of the following mechanisms may contribute to treatment of TTRamyloidosis using the above antibodies: antibody-mediated inhibition ofTTR aggregation and fibril formation, antibody-mediated stabilization ofnon-toxic conformations of TTR (e.g., tetrameric forms), orantibody-mediated clearance of aggregated TTR, oligomeric TTR, ormonomeric TTR. Antibody-drug conjugates can have additional mechanismsof action determined by the conjugated moiety.

Antibodies are administered in an effective regime meaning a dosage,route of administration and frequency of administration that delays theonset, reduces the severity, inhibits further deterioration, and/orameliorates at least one sign or symptom of a disorder being treated. Ifa patient is already suffering from a disorder, the regime can bereferred to as a therapeutically effective regime. If the patient is atelevated risk of the disorder relative to the general population but isnot yet experiencing symptoms, the regime can be referred to as aprophylactically effective regime. In some instances, therapeutic orprophylactic efficacy can be observed in an individual patient relativeto historical controls or past experience in the same patient. In otherinstances, therapeutic or prophylactic efficacy can be demonstrated in apreclinical or clinical trial in a population of treated patientsrelative to a control population of untreated patients.

The frequency of administration depends on the half-life of the antibodyin the circulation, the condition of the patient and the route ofadministration among other factors. The frequency can be daily, weekly,monthly, quarterly, or at irregular intervals in response to changes inthe patient's condition or progression of the disorder being treated. Anexemplary frequency for intravenous administration is between weekly andquarterly over a continuous cause of treatment, although more or lessfrequent dosing is also possible. For subcutaneous administration, anexemplary dosing frequency is daily to monthly, although more or lessfrequent dosing is also possible.

The number of dosages administered depends on whether the disorder isacute or chronic and the response of the disorder to the treatment. Foracute disorders or acute exacerbations of a chronic disorder, between 1and 10 doses are often sufficient. Sometimes a single bolus dose,optionally in divided form, is sufficient for an acute disorder or acuteexacerbation of a chronic disorder. Treatment can be repeated forrecurrence of an acute disorder or acute exacerbation. For chronicdisorders, an antibody can be administered at regular intervals, e.g.,weekly, fortnightly, monthly, quarterly, every six months for at least1, 5 or 10 years, or the life of the patient.

VI. Pharmaceutical Compositions and Methods of Use

Provided herein are several methods of diagnosing, monitoring, treatingor effecting prophylaxis of diseases or conditions mediated at least inpart by transthyretin (TTR), and particularly by monomeric, misfolded,aggregated, or fibril forms of TTR (e.g., TTR amyloidosis). Examples ofsuch diseases include familial TTR amyloidoses, such as familial amyloidcardiomyopathy (FAC) or cardiomyopathy or hypertrophy in athletes orothers undergoing extreme aerobic exercise, familial amyloidpolyneuropathy (FAP), or central nervous system selective amyloidosis(CNSA), and sporadic TTR amyloidoses, such as senile systemicamyloidosis (SSA) or senile cardiac amyloidosis (SCA). TTR amyloidosiscan also be associated as a cause or result of various diseases andconditions characterized by tissue or organ degeneration or trauma.Accumulation of TTR deposits contributes to organ or tissue dysfunctionassociated with the disease or condition. An example of such a conditionamenable to treatment or prophylaxis with the present agents and methodsis spinal stenosis (Westermark et al., Upsala J. Medical Sciences 119,223-238 (2014) and Yanagisawa et al., Modern Pathology 28, 201-207(2015). Another disease likewise amenable to treatment or prophylaxis isosteoarthritis (Takanashi et al., Amyloid 20, 151-155 (2013), Gu et al.,Biomed & Biotechnol. 15, 92-99; Takinami et al., Biomarker Insights 8,85-95 (2014); Akasaki et al., Arthritis Rheumatol. 67, 2097-2107 (2015).Another disease likewise amenable to treatment or prophylaxis isrheumatoid arthritis (Clement et al., JCI Insight 1 epublish (2016).Another disease amenable to treatment or prophylaxis is juvenileidiopathic arthritis (Sharma et al., PLOSone 9, 1-12 (2014). Anotherdisease amenable to treatment or prophylaxis is age related maculardegeneration (wet or dry). Another class of conditions likewise amenableto treatment or prophylaxis are ligament and tendon disorders, such asdisorders of the rotator cuff (Sueyoshi et al., Human Pathol. 42,1259-64 (2011).

Antibodies described above can be incorporated into a pharmaceuticalcomposition for use treatment or prophylaxis of any of the abovediseases and conditions. In general, an antibody or pharmaceuticalcomposition containing an antibody is administered to a subject in needthereof. Patients amenable to treatment include individuals at risk ofTTR amyloidosis but not showing symptoms, as well as patients presentlyshowing symptoms. Some patients can be treated during the prodromalstage of TTR amyloidosis.

Individuals suffering from TTR amyloidosis can sometimes be recognizedfrom the clinical manifestations of TTR amyloidosis, including one ormore of the following: (1) family history of neuropathic disease,especially associated with heart failure; (2) neuropathic pain orprogressive sensory disturbances of unknown etiology; (3) carpal tunnelsyndrome without obvious cause, particularly if it is bilateral andrequires surgical release; (4) gastrointestinal motility disturbances orautonomic nerve dysfunction of unknown etiology (e.g., erectiledysfunction, orthostatic hypotension, neurogenic gladder); (5) cardiacdisease characterized by thickened ventricular walls in the absence ofhypertension; (6) advanced atrio-ventricular block of unknown origin,particularly when accompanied by a thickened heart; and (6) vitreousbody inclusions of the cotton-wool type. See Ando et al., OrphanetJournal of Rare Diseases 8:31 (2013). Definitive diagnosis of TTRamyloidosis, however, typically relies on target organ biopsies,followed by histological staining of the excised tissue with theamyloid-specific dye, Congo red. If a positive test for amyloid isobserved, immunohistochemical staining for TTR is subsequently performedto ensure that the precursor protein responsible for amyloid formationis indeed TTR. For familial forms of the diseases, demonstration of amutation in the gene encoding TTR is then needed before a definitivediagnosis can be made.

The identification of the subject can occur in a clinical setting, orelsewhere, such as in the subject's home, for example, through thesubject's own use of a self-testing kit. For example, the subject can beidentified based on various symptoms such as peripheral neuropathy(sensory and motor), autonomic neuropathy, gastrointestinal impairment,cardiomyopathy, nephropathy, or ocular deposition. See Ando et al.,Orphanet Journal of Rare Diseases 8:31 (2013). The subject can also beidentified by increased levels of non-native forms of TTR in plasmasamples from the subject compared to control samples, as disclosed inthe examples.

As warranted by family history, genetic testing, or medical screeningfor TTR amyloidosis, treatment can begin at any age (e.g., 20, 30, 40,50, 60, or 70 years of age). Treatment typically entails multipledosages over a period of time and can be monitored by assaying antibodyor activated T-cell or B-cell responses to a therapeutic agent (e.g., atruncated form of TTR comprising amino acid residues 89-97) over time.If the response falls, a booster dosage is indicated.

In prophylactic applications, an antibody or a pharmaceuticalcomposition of the same is administered to a subject susceptible to, orotherwise at risk of a disease (e.g., TTR amyloidosis) in a regime(dose, frequency and route of administration) effective to reduce therisk, lessen the severity, or delay the onset of at least one sign orsymptom of the disease. In therapeutic applications, an antibody orimmunogen to induce an antibody is administered to a subject suspectedof, or already suffering from a disease (e.g., TTR amyloidosis) in aregime (dose, frequency and route of administration) effective toameliorate or at least inhibit further deterioration of at least onesign or symptom of the disease.

A regime is considered therapeutically or prophylactically effective ifan individual treated subject achieves an outcome more favorable thanthe mean outcome in a control population of comparable subjects nottreated by methods disclosed herein, or if a more favorable outcome isdemonstrated for a regime in treated subjects versus control subjects ina controlled clinical trial (e.g., a phase II, phase II/III, or phaseIII trial) or an animal model at the p<0.05 or 0.01 or even 0.001 level.

An effective regime of an antibody can be used for, e.g., inhibiting orreducing aggregation of TTR in a subject having or at risk of acondition associated with TTR accumulation; inhibiting or reducing TTRfibril formation in a subject having or at risk of a conditionassociated with TTR accumulation; reducing or clearing TTR deposits oraggregated TTR in a subject having or at risk of a condition associatedwith TTR accumulation; stabilizing non-toxic conformations of TTR in asubject having or at risk of a condition associated with TTRaccumulation; inhibiting toxic effects of TTR aggregates, fibrils ordeposits in a subject having or at risk of a condition associated withTTR accumulation; diagnosing the presence or absence of TTR amyloidaccumulation in a tissue suspected of comprising the amyloidaccumulation; determining a level of TTR deposits in a subject bydetecting the presence of bound antibody in the subject followingadministration of the antibody; detecting the presence of monomeric,misfolded, aggregated, or fibril forms of TTR in a subject; monitoringand evaluating the efficacy of therapeutic agents being used to treatpatients diagnosed with a TTR amyloidosis; inducing an immune responsecomprising antibodies to TTR in a subject; delaying the onset of acondition associated with TTR amyloid accumulation in a subject; ortreating or effecting prophylaxis of a TTR amyloidosis in a patient.

Effective doses vary depending on many different factors, such as meansof administration, target site, physiological state of the subject,whether the subject is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.

An exemplary dose range for antibodies can be from about 0.1-20, or0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-1500 mgas a fixed dosage. The dosage depends on the condition of the patientand response to prior treatment, if any, whether the treatment isprophylactic or therapeutic and whether the disorder is acute orchronic, among other factors.

Antibody can be administered in such doses daily, on alternative days,weekly, fortnightly, monthly, quarterly, or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple doses over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimesentail administration once per every two weeks or once a month or onceevery 3 to 6 months.

Antibodies can be administered via a peripheral route. Routes ofadministration include topical, intravenous, oral, subcutaneous,intraarterial, intracranial, intrathecal, intraperitoneal, intranasal orintramuscular. Routes for administration of antibodies can beintravenous or subcutaneous. Intravenous administration can be, forexample, by infusion over a period such as 30-90 min. This type ofinjection is most typically performed in the arm or leg muscles. In somemethods, agents are injected directly into a particular tissue wheredeposits have accumulated, for example intracranial injection.

Pharmaceutical compositions for parenteral administration can be sterileand substantially isotonic (250-350 mOsm/kg water) and manufacturedunder GMP conditions. Pharmaceutical compositions can be provided inunit dose form (i.e., the dose for a single administration).Pharmaceutical compositions can be formulated using one or morephysiologically acceptable carriers, diluents, excipients orauxiliaries. The formulation depends on the route of administrationchosen. For injection, antibodies can be formulated in aqueoussolutions, e.g., in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological saline or acetate buffer(to reduce discomfort at the site of injection). The solution cancontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively antibodies can be in lyophilized formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

The regimes can be administered in combination with another agenteffective in treatment or prophylaxis of the disease being treated. Suchagents can include siRNA to inhibit expression of TTR or Vyndaqel, astabilizer of TTR in tetramer formation.

After treatment, the subject's condition can be evaluated to determinethe progress or efficacy of such treatment. Such methods preferably testfor changes in TTR amyloid levels or levels of non-native forms of TTR.For example, TTR amyloid levels may be evaluated to determineimprovement relative to the subject's TTR amyloid levels undercomparable circumstances prior to treatment. The subject's TTR amyloidlevels can also be compared with control populations under comparablecircumstances. The control populations can be similarly afflicted,untreated subjects or normal untreated subjects (among other controlsubjects). Improvement relative to similarly afflicted, untreatedsubjects or levels approaching or reaching the levels in untreatednormal subjects indicates a positive response to treatment.

TTR amyloid levels can be measured by a number of methods, includingimaging techniques. Examples of suitable imaging techniques include PETscanning with radiolabeled TTR of fragments thereof, TTR antibodies orfragments thereof, Congo-red-based amyloid imaging agents, such as,e.g., PIB (US 2011/0008255), amyloid-imaging peptide p31(Biodistribution of amyloid-imaging peptide, p31, correlates withamyloid quantitation based on Congo red tissue staining, Wall et al.,Abstract No. 1573, 2011 ISNM Annual Meeting), and other PET labels.Levels of non-native forms of TTR can be measured, for example, byperforming SDS-PAGE/Western blot or Meso Scale Discovery plate assayswith the antibodies disclosed herein on plasma samples or biopsy samplesfrom a subject and comparing to control samples, as described in theexamples.

A. Diagnostics and Monitoring Methods

Also provided are methods of detecting an immune response against TTR ina patient suffering from or susceptible to diseases associated with TTRdeposition or pathogenic forms of TTR (e.g., monomeric, misfolded,aggregated, or fibril forms of TTR). The methods can be used to monitora course of therapeutic and prophylactic treatment with the agentsprovided herein. The antibody profile following passive immunizationtypically shows an immediate peak in antibody concentration followed byan exponential decay. Without a further dose, the decay approachespretreatment levels within a period of days to months depending on thehalf-life of the antibody administered. For example, the half-life ofsome human antibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to TTR in thesubject is made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dose of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other subjects. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one or, preferably, two standarddeviations of the reference value in a population of subjects benefitingfrom treatment) administration of an additional dose of antibody isindicated.

Also provided are methods of detecting monomeric, misfolded, aggregated,or fibril forms of TTR in a subject, for example, by measuring TTRamyloid or pathogenic forms of TTR (e.g., monomeric, misfolded,aggregated, or fibril forms of TTR) in a sample from a subject or by invivo imaging of TTR in a subject. Such methods are useful to diagnose orconfirm diagnosis of diseases associated with such pathogenic forms ofTTR (e.g., TTR amyloidosis), or susceptibility thereto. The methods canalso be used on asymptomatic subjects. The presence of monomeric,misfolded, aggregated, or fibril forms of TTR indicates susceptibilityto future symptomatic disease. The methods are also useful formonitoring disease progression and/or response to treatment in subjectswho have been previously diagnosed with a TTR amyloidosis.

Biological samples obtained from a subject having, suspected of having,or at risk of having a TTR amyloidosis can be contacted with theantibodies disclosed herein to assess the presence of monomeric,misfolded, aggregated, or fibril forms of TTR. For example, levels ofmonomeric, misfolded, aggregated, or fibril forms of TTR in suchsubjects may be compared to those present in healthy subjects.Alternatively, levels of TTR amyloid or pathogenic forms of TTR (e.g.,monomeric, misfolded, aggregated, or fibril forms of TTR) in suchsubjects receiving treatment for the disease may be compared to those ofsubjects who have not been treated for a TTR amyloidosis. Some suchtests involve a biopsy of tissue obtained from such subjects. ELISAassays may also be useful methods, for example, for assessing levels ofmonomeric, misfolded, aggregated, or fibril forms of TTR in fluidsamples. Some such ELISA assays involve anti-TTR antibodies thatpreferentially bind monomeric, misfolded, aggregated, or fibril forms ofTTR relative to normal tetrameric forms of TTR.

The in vivo imaging methods can work by administering a reagent, such asantibody that binds to monomeric, misfolded, aggregated, or fibril formsof TTR in the subject, and then detecting the reagent after it hasbound. Such antibodies typically bind to an epitope within residues89-97 of TTR. If desired, the clearing response can be avoided by usingantibody fragments lacking a full length constant region, such as Fabs.In some methods, the same antibody can serve as both a treatment anddiagnostic reagent.

Diagnostic reagents can be administered by intravenous injection intothe body of the subject, or via other routes deemed reasonable. The doseof reagent should be within the same ranges as for treatment methods.Typically, the reagent is labeled, although in some methods, the primaryreagent with affinity for monomeric, misfolded, aggregated, or fibrilforms of TTR is unlabeled and a secondary labeling agent is used to bindto the primary reagent. The choice of label depends on the means ofdetection. For example, a fluorescent label is suitable for opticaldetection. Use of paramagnetic labels is suitable for tomographicdetection without surgical intervention. Radioactive labels can also bedetected using PET or SPECT.

Diagnosis is performed by comparing the number, size, and/or intensityof labeled loci to corresponding base line values. The base line valuescan represent the mean levels in a population of undiseased individuals.Base line values can also represent previous levels determined in thesame subject. For example, base line values can be determined in asubject before beginning treatment, and measured values thereaftercompared with the base line values. A decrease in values relative tobase line generally signals a positive response to treatment.

IX. Kits

The invention further provides kits (e.g., containers) comprising thehumanized 6C1 antibodies disclosed herein and related materials, such asinstructions for use (e.g., package insert). The instructions for usemay contain, for example, instructions for administration of theantibodies and optionally one or more additional agents. The containersof antibodies may be unit doses, bulk packages (e.g., multi-dosepackages), or sub-unit doses.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products

Kits can also include a second container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It can also include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

X. Other Applications

The antibodies can be used for detecting monomeric, misfolded,aggregated, or fibril forms of transthyretin (TTR), or fragmentsthereof, in the context of clinical diagnosis or treatment or inresearch. For example, the antibodies can be used to detect the presenceof monomeric, misfolded, aggregated, or fibril forms of TTR in abiological sample as an indication that the biological sample comprisesTTR amyloid deposits. Binding of the antibodies to the biological samplecan be compared to binding of the antibodies to a control sample. Thecontrol sample and the biological sample can comprise cells of the sametissue origin. Control samples and biological samples can be obtainedfrom the same individual or different individuals and on the sameoccasion or on different occasions. If desired, multiple biologicalsamples and multiple control samples are evaluated on multiple occasionsto protect against random variation independent of the differencesbetween the samples. A direct comparison can then be made between thebiological sample(s) and the control sample(s) to determine whetherantibody binding (i.e., the presence of monomeric, misfolded,aggregated, or fibril forms of TTR) to the biological sample(s) isincreased, decreased, or the same relative to antibody binding to thecontrol sample(s). Increased binding of the antibody to the biologicalsample(s) relative to the control sample(s) indicates the presence ofmonomeric, misfolded, aggregated, or fibril forms of TTR in thebiological sample(s). In some instances, the increased antibody bindingis statistically significant. Optionally, antibody binding to thebiological sample is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,10-fold, 20-fold, or 100-fold higher than antibody binding to thecontrol sample.

In addition, the antibodies can be used to detect the presence ofmonomeric, misfolded, aggregated, or fibril forms of TTR in a biologicalsample to monitor and evaluate the efficacy of a therapeutic agent beingused to treat a patient diagnosed with a TTR amyloidosis. A biologicalsample from a patient diagnosed with a TTR amyloidosis is evaluated toestablish a baseline for the binding of the antibodies to the sample(i.e., a baseline for the presence of the monomeric, misfolded,aggregated, or fibril forms of TTR in the sample) before commencingtherapy with the therapeutic agent. In some instances, multiplebiological samples from the patient are evaluated on multiple occasionsto establish both a baseline and measure of random variation independentof treatment. A therapeutic agent is then administered in a regime. Theregime may include multiple administrations of the agent over a periodof time. Optionally, binding of the antibodies (i.e., presence ofmonomeric, misfolded, aggregated, or fibril forms of TTR) is evaluatedon multiple occasions in multiple biological samples from the patient,both to establish a measure of random variation and to show a trend inresponse to immunotherapy. The various assessments of antibody bindingto the biological samples are then compared. If only two assessments aremade, a direct comparison can be made between the two assessments todetermine whether antibody binding (i.e., presence of monomeric,misfolded, aggregated, or fibril forms of TTR) has increased, decreased,or remained the same between the two assessments. If more than twomeasurements are made, the measurements can be analyzed as a time coursestarting before treatment with the therapeutic agent and proceedingthrough the course of therapy. In patients for whom antibody binding tobiological samples has decreased (i.e., the presence of monomeric,misfolded, aggregated, or fibril forms of TTR), it can be concluded thatthe therapeutic agent was effective in treating the TTR amyloidosis inthe patient. The decrease in antibody binding can be statisticallysignificant. Optionally, binding decreases by at least 1%, 2%, 3%, 4%,5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.Assessment of antibody binding can be made in conjunction with assessingother signs and symptoms of TTR amyloidosis.

The antibodies can also be used as research reagents for laboratoryresearch in detecting monomeric, misfolded, aggregated, or fibril formsof TTR, or fragments thereof. In such uses, antibodies can be labeledwith fluorescent molecules, spin-labeled molecules, enzymes, orradioisotopes, and can be provided in the form of kit with all thenecessary reagents to perform the detection assay. The antibodies canalso be used to purify monomeric, misfolded, aggregated, or fibril formsof TTR, or binding partners of monomeric, misfolded, aggregated, orfibril forms of TTR, e.g., by affinity chromatography.

The antibodies can also be used for inhibiting or reducing aggregationof TTR, inhibiting or reducing TTR fibril formation, reducing orclearing TTR deposits or TTR aggregates, or stabilizing non-toxicconformations of TTR in a biological sample. The biological sample cancomprise, for example, blood, serum, plasma, or tissue (e.g., tissuefrom the heart, peripheral nervous system, autonomic nervous system,kidneys, eyes, or gastrointestinal tract). In some instances, TTRaggregation, TTR fibril formation, or TTR deposits are inhibited orreduced by at least 10%, 20%, 25%, 30%, 40%, 50%, or 75%, (e.g., 10%-75%or 30%-70%). Assays for detecting fibril formation are describedelsewhere herein. See also US 2014/0056904.

Antibody 6C1 has been deposited subject to the Budapest Treaty underaccession number PTA-124077 on Apr. 4, 2017 at the American Type CultureCollection 10801 University Boulevard Manassas, Va. 20110 USA. Allrestrictions imposed by the depositor on the availability to the publicof the deposited material will be irrevocably removed on the granting ofa patent except as permitted under 37 CFR 1.808(b).

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example 1. Identification of Mis-TTR Monoclonal Antibodies

Conformationally-specific monoclonal antibodies against monomeric,mis-folded, fibril, or aggregated forms of TTR (mis-TTR) were generated,screened, expressed, and purified as described in Materials and Methods(a-d). In order to generate mis-TTR monoclonal antibodies, the crystalstructure of human tetrameric TTR was examined to find regions of theprotein that are buried in the tetramer, but exposed upon dissociationof the tetramer into its monomeric subunits. The region identified wasresidues 89-97 (EHAEVVFTA) (SEQ ID NO:42) located within the F strand ofTTR and sequestered at the dimer interface of the tetrameric protein. ABLAST search of the protein database did not reveal any other humanproteins possessing this sequence.

A peptide comprising this sequence (ggEHAEVVFTAggkg) (SEQ ID NO:43), wassynthesized. Capitalized letters represent residues 89-97 of TTR. Lowercase letters represent additional linker residues added to increase thesolubility of the antigenic peptide and to establish the 9 amino acidfragment as an internal sequence. This peptide was linked to apoly-lysine dendritic core, generating a multiple antigenic peptideimmunogen (TTR-MAP) comprising a core of lysine residues with multiplebranches linked to the TTR 89-97 peptide. The antibodies listed in Table2 were generated against TTR-MAP.

In addition to this multiple antigenic peptide, two other immunogenscontaining the same TTR fragment were generated by covalently linkingsimilar TTR 89-97 peptides (Ac-cggEHAEVVFTA-amide (SEQ ID NO:44) andAc-EHAEVVFTAcgg-amide) (SEQ ID NO:45) via the N- and C-terminal cysteineresidues to keyhole limpet hemocyanin (TTR89-97-N-KLH andTTR89-97-C-KLH).

Following antibody generation, screening, expression, and purification,detailed binding kinetic parameters (association rate (k_(a)),dissociation rate (k_(d)), and binding affinity constant (K_(D))) weredetermined for lead mis-TTR antibodies by Surface Plasmon Resonance(SPR) for recombinant human TTR F87M/L110M, as shown in Table 2.Anti-mouse IgG (GE Healthcare) was immobilized on a sensor chip C5(lacking dextran chains) via amine coupling following the instructionsprovided in the GE Healthcare anti-mouse kit, and mis-TTR mAbs werecaptured to a level to ensure a maximum binding of analyte of 30-50 RU.Various concentrations of analyte (recombinant human TTR F87M/L110M)were passed over the captured ligand at 30 μl/min in running buffer(HBS+0.05% P-20, 1 mg/mL BSA) in 3-fold dilutions. For eachconcentration, the reaction proceeded for a time frame allowing for thehigher analyte concentrations to reach equilibrium during association,as well as at least 10% of signal to decay during dissociation. At leastone concentration (not the highest or lowest) was run in duplicate.Concentration ranges of analyte were selected based on preliminaryexperimentation to span at least 10-fold above K_(D) to 10-fold belowK_(D).

The results of SPR analysis of lead mis-TTR mAbs is shown in Table 2below.

TABLE 2 SPR Analysis of Lead mis-TTR Antibodies Binding to Human TTR(F87M/L110M) mAb k_(a)(1/Ms) k_(d)(1/s) K_(D)(M) R_(max) 9D5 2.715E+44.930E−4 1.816E−8 31.55 14G8 2.880E+4 5.358E−4 1.861E−8 27.13 5A16.107E+4 4.693E−4 7.684E−9 30.98 6C1 4.607E+4 4.151E−4 9.010E−9 26.32

Example 2. Binding of Mis-TTR Antibodies to TTR Antigen

Four lead mis-TTR mAbs (9D5, 14G8, 6C1, and 5A1) were assayed by ELISAat concentrations ranging from 0.31 to 2.5 μg/ml using bothpH4.0-treated TTR (pH4-TTR) and native TTR as the coating antigen. TTRantigen preparation and ELISA protocols are described elsewhere inMaterials and Methods (e-g).

The resulting binding curves and tabulated K_(d) and B_(max) values areshown in FIG. 3 and Table 3 below. The results in FIG. 3 are presentedin arbitrary units (a.u.) on the y-axis. All mAbs showed significantbinding to pH4-TTR with K_(d) values ranging from 16 nM (6C1) to 282 nM(9D5). B_(max) values for binding to pH4-TTR ranged from a low of 0.65a.u. (14G8) to a high of 2.02 (9D5). In contrast to the binding topH4-TTR, none of the antibodies showed significant binding to nativeTTR, indicating that all TTR antibodies generated were specific fornon-native forms of TTR.

TABLE 3 ELISA Analysis of Lead mis-TTR Antibodies Binding to pH4-TTR mAbK_(d) (nM) B_(max) (a.u.) 9D5 282 2.02 14G8 108 0.65 6C1 16 1.07 5A1 231.61

Example 3. Analysis of Mis-TTR Antibodies by SDS-PAGE and Native-PAGE

9D5 and 14G8 were analyzed by SDS-PAGE/Western to demonstratespecificity of binding toward monomeric/denatured forms of TTR versusnative, non-denatured TTR. SDS-PAGE, Native-PAGE, and Western Blotprotocols are described elsewhere in the Methods and Materials (h-j).

Non-denatured TTR or pH4-TTR was run on an SDS-PAGE gel alongsideheat-denatured TTR and heat-denatured pH4-TTR. After electrophoresis,the gel was Western blotted onto nitrocellulose and stained with TTRmAbs 9D5 and 14G8. Both antibodies only recognized TTR when it wastreated at pH4 or when TTR or pH4-TTR was first heat-denatured prior toSDS-PAGE. These 9D5 and 14G8 thus show a specificity for TTR conformersgenerated either by denaturation of TTR or by treatment of TTR at pH4.

6C1 and 5A1 along with total TTR mAbs (7G7, 8C3) and the commerciallyavailable Sigma polyclonal antibody were also analyzed bySDS-PAGE/Western. Each blot contained stained molecular weight markers,non-denatured TTR, and pH4-TTR.

The stained SDS-PAGE gel showed that the major species present in thenon-denatured TTR sample was an ˜38 kDa dimer. In contrast, the majorcomponent present in the pH4-TTR sample ran as an ˜35 kDa dimer with asmall amount of dimer of an ˜15 kDa monomer. This dimer ran as aslightly smaller protein than the dimer present in the non-denatured TTRsample, indicating a conformational difference between these two TTRdimer species.

The Western blots of TTR and pH4-TTR using the four mis-TTR antibodiesshowed that these mAbs do not recognize non-denatured TTR, but do bindto both the denatured monomer and dimer present in the pH4-TTR sample.Thus, the four mis-TTR mAbs (9D5, 14G8, 6C1, and 5A1) show similarspecificities for non-native conformations of TTR when analyzed bySDS-PAGE/Westerns.

In contrast to the four mis-TTR mAbs, the two TTR control mAbs, 7G7 and8C3 generated through immunization of mice with intact TTR recognizedall TTR species present in the TTR and pH4-TTR samples, includingtetrameric TTR species. Thus unlike the mis-TTR mAbs, these control mAbsbind TTR but with no conformational specificity. The Sigma polyclonalantibody behaved similarly to the 7G7 and 8C3 control mAbs.

TTR and pH4-TTR were also run on a native gel to see if the four mis-TTRmAbs were capable of showing conformation specificity undernon-denaturing gel conditions. On a stained native PAGE gel, TTR ran asan ˜35 kDa native dimer with a small amount of tetramer. In contrast,pH4-TTR ran primarily as a high molecular-weight smear with a traceamount of the ˜35 kDa dimer. The non-specific Sigma polyclonal antibodyrecognized all TTR species present in both the TTR and the pH4-TTRsample. In contrast, 9D5 only recognized the high molecular weight TTRspecies present in the pH4-TTR sample. As observed in theSDS-PAGE/Western study, 9D5 did not recognize any of the native TTRspecies.

All four mis-TTR mAbs were subsequently analyzed by native-PAGE/Westernblot. As expected and similar to 9D5, the other mis-TTR mAbs, 14G8, 6C1,and 5A1, specifically bound to the high molecular weight non-nativeforms of TTR present in the pH4-TTR sample. None of these antibodiesrecognized the ˜35 kDa native TTR dimer. These results indicate that thefour mis-TTR mAbs behave similarly and recognize only non-native TTRspecies that are conformationally distinct from native TTR.

Example 4. Inhibition of TTR Fiber Formation by Mis-TTR Antibodies

TTR-Y78F is a TTR variant containing a point mutation at position 78 inthe protein sequence that destabilizes the TTR tetramer. With time andunder mildly acidic conditions, this TTR variant dissociates into itsmonomeric subunits which can then go on to aggregate and form fiberscapable of binding to thioflavin-T. The extent of fiber formation canthus be monitored by measuring thioflavin-T fluorescence at 480 nm.Introduction of a mis-TTR antibody specific for dissociated TTR monomersor aggregates would prevent the assembly of TTR fibers resulting in adecrease in thioflavin-T fluorescence relative to a no-antibody controlreaction. Protocols for examining inhibition of TTR fiber formation aredescribed elsewhere in the Materials and Methods (k).

All four mis-TTR antibodies strongly inhibited the formation ofthioflavin-T reactive TTR-Y78F fibers relative to the isotypecontrol(the results are shown in FIG. 4 and are presented in arbitraryunits (a.u.) on the y-axis). Mis-TTR antibody 5A1 almost completelyinhibited fiber formation. These results are consistent with the notionthat mis-TTR antibodies bind monomeric and/or aggregated forms of TTR,thereby preventing the formation of TTR fibers.

Table 4 summarizes the characterization data obtained for the set of 4mis-TTR antibodies (9D5, 14G8, 6C1, and 5A1) that showed goodconformational selectivity for non-native forms of TTR. These antibodieshad affinities (K_(D)) for pH4-TTR ranging from 14.5 nM (6C1) to 257 nM(9D5) and B_(max) values ranging from 0.65 a.u. (14G8) to 2.02 (9D5).None of these antibodies recognized native TTR, but did bind to pH4-TTRon an SDS-PAGE/Western and to the high molecular weight TTR aggregateson a native-PAGE/Western. These antibodies also inhibited the formationof TTR fibrils in the fibril formation assay using Thio-T as theread-out.

TABLE 4 mis-TTR-Y78F mAb Characterization Summary Table Sandwich ELISA(pH 4-TTR) Western Blot B_(max) SDS-PAGE Native % Inh. Clone K_(D)(OD₄₅₀ (pH 4- (HMW- Fibrils ID (nM) a.u.) (TTR) TTR) TTR) (Thio-T) 9D5257 2.02 − +++ +++ 83 14G8 98.7 0.65 − +++ ++ 65 6C1 14.6 1.07 − +++ +++72 5A1 21.3 1.61 − +++ +++ 100

TTR-V122I is a TTR variant containing a single point mutation atposition 122 that destabilizes the tetramer. Fibril formation wasassociated with an increase in ThT fluorescence Increasing 14G8 mAbconcentrations caused a monotonic decrease in ThT fluorescenceindicating a substoichiometric inhibition of TTR fibrillation(IC₅₀=0.028±0.009 mg/mL; n=3; FIG. 4B and Table 4a). The isotype controlmAb did not cause inhibition of TTR fibrillation (FIG. 4C), thusdemonstrating the specificity of 14G8 mediated inhibition.

Comparable substoichiometric IC₅₀ values determined for 5A1 and 6C1(Table 4a) suggested analogous mechanisms of fibril inhibition for eachof these mis-TTR mAbs. In contrast, 9D5 unexpectedly failed to inhibitTTR-V122I fibril formation, despite showing similar specificity andaffinity for non-native TTR. It remains to be explored whether 9D5 ismore sensitive to the assay conditions used.

TABLE 4a mis-TTR-V122I mAb Characterization Summary Table Antibody IC₅₀± SD (mg/mL) 9D5 No inhibition 14G8 0.028 ± 0.009 6C1 0.048 ± 0.059 5A10.015 ± 0.02  EG 27/1 No inhibition

Example 5. Immunohistochemical (IHC) Characterization of ATTR TissueUsing Mis-TTR mAbs

The lead mis-TTR mAbs raised to the TTR 89-97 fragment of thetransthyretin protein were immunohistochemically tested on fresh frozenand paraffin processed tissue from confirmed TTR cardiac amyloidosispatients. Protocols for obtaining and preparing cardiac tissue samples,immunohistochemistry (IHC), and image analysis, are provided elsewherein the Materials and Methods (l-o). The antibodies used for IHC aredescribed in Table 5.

TABLE 5 Antibodies Used for Immunohistochemical Characterization StainAntibody Cardiac Antibody Type Vendor Tissue Concentration 14G8 mis-TTRProthena Yes 0.5 μg/mL Biosciences 9D5 mis-TTR Prothena Yes 0.5 μg/mLBiosciences 6C1 mis-TTR Prothena Yes 0.5 μg/mL Biosciences 5A1 mis-TTRProthena Yes 0.5 μg/mL Biosciences 7G7 TTR Prothena Yes 0.5 μg/mLBiosciences 6F10 Isotype Prothena No 0.5 μg/mL Control BiosciencesPrealbumin TTR Dako North Yes 1:2,000 & (A0002) America 1:20,000 KappaLight LC-κ Dako North No 1:8,000 Chains America (A0191) Lambda LightLC-λ Dako North No 1:8,000 Chains America (A0193) Amyloid A AA DakoNorth No 1:8,000 (M0759) America

Cardiac tissue samples were obtained from patients with confirmeddiagnoses of ATTR mutations. Demographics for cases examinedimmunohistochemically were as follows and are summarized in Table 6:FAC=familial amyloidotic cardiomyopathy; FAP=familial amyloidoticpolyneuropathy; 1° AL=light-chain amyloidosis;ATTR=transthyretin-mediated amyloidosis; Unk=Unknown

TABLE 6 Immunohistochemical Staining of Cardiac Tissue Samples withmis-TTR Antibodies TTR Stained with TTR Case Diagnosis Mutations FormatAntibodies? Patient 1 FAC Ileu122 Frozen Yes Patient 2 FAP Wild typeFrozen Yes Patient 3 FAP 84Ser Frozen Yes Patient 4 FAP 84Ser Frozen YesPatient 5 1° AL — Frozen No Patient 6 1° AL — Frozen No Patient 7 ATTR10Arg Frozen Yes Patient 8 ATTR V122I Frozen Yes Patient H1 ATTRVal122Ile FFPE Yes Patient H2 ATTR Thr60Ala FFPE Yes Patient H3 ATTRThr49Ala FFPE Yes Patient H4 ATTR Ile84Ser FFPE Yes Patient H5 Unk.Senile FFPE Yes Cardiac Patient H6 ATTR Ile84Ser FFPE Yes

Mouse monoclonal antibodies (mis-TTR mAbs) raised to the 89-97 fragmentof the transthyretin protein were immunohistochemically tested on freshfrozen and paraffin processed tissue from confirmed TTR cardiacamyloidosis patients. Each mis-TTR antibody showed immunoreactivity onATTR cardiac tissue. Dark staining was observed in deposits throughoutthe myocardium and the vasculature. When immunoreactivity was comparedto staining with Congo Red of Thioflavin-T, the majority of theimmunoreactivity in the tissue showed high congruence with Congo redbirefringence and Thioflavin T-positive staining. This confirms the betapleated sheet nature of the TTR amyloid deposited in this tissue. Thesemis-TTR antibodies also detected pre-amyloid TTR, which were localizedto areas of the myocardium that were TTR-immunopositive but Congo red orThioflavin T-negative. Both the IgG-isotype control antibody and primaryantibody omission sections were negative for staining across all tissuestested. Antibodies reactive toward other amyloidogenic proteins (lambdaand kappa light chains or amyloid A) were non-reactive toward the ATTRcardiac tissue used in this analysis, indicating that deposits werespecifically TTR in nature.

The staining pattern of mis-TTR antibodies were compared to thatobtained with a well characterized commercial TTR reference antibody(prealbumin, A0002; Dako; Carpinteria, Calif.). The DAKO referenceantibody stained the diseased myocardium in the same areas as themis-TTR antibodies, but produced a more diffuse staining pattern. TheDAKO reference antibody did not stain the congophillic TTR amyloiddeposits present on the vasculature as strongly as the mis-TTRantibodies.

The mis-TTR antibodies did not stain normal, non-disease tissue.Furthermore, as expected, staining with an isotype control antibody,6F10 was also negative.

To determine if the reactivity of mis-TTR antibodies was specific forTTR deposits, cross reactivity of these antibodies toward cardiac tissuederived from patients diagnosed with primary AL amyloidosis wasexamined. As expected, no staining of AL amyloid tissue was observed,confirming that TTR antibodies react specifically toward ATTR diseasedtissue.

Cardiac tissue from patients with confirmed diagnoses of senile systemicamyloidosis or from patients with confirmed FAC, or FAP caused by pointmutations in the TTR gene also stained positively with 14G8, 9D5, 6C1,and 5A1. These results indicate that mis-TTR antibodies have the abilityto recognize TTR deposits in cardiac tissue regardless of the ATTRgenotype.

Other non-cardiac tissues known to express TTR were also examined forstaining by 14G8, 9D5, 6C1, and 5A1 and compared to the stainingobtained using the DAKO reference antibody. As expected, the liver,pancreas and choroid plexus all stained positively for TTR using theDako reference antibody. In contrast, mis-TTR antibodies only stainedthe pancreatic alpha cells located in the islets of Langerhans and thechoroid plexus, suggesting that some of the TTR localized to theseorgans are conformationally distinct from TTR expressed in the liver.The lack of mis-TTR mAb immunoreactivity in the liver suggests that thelarge amount of TTR expressed there is primarily tetrameric, native TTRand does not have the exposed mis-TTR epitope.

Example 6. Analysis of ATTR Vs Normal Human Plasma by SDS-PAGE/WesternBlot and by Meso Scale Discovery (MSD) Plate Assay

Six plasma samples from patients confirmed for V30M ATTR (Sample #11,#12, #15, #18, #19, #20) and 6 samples (#21, #22, #23, #24, #25, #27)from normal subjects were obtained from M. Saraiva (Porto University,Portugal). Sample #C6 was a normal human serum sample obtained from acommercial source (BioreclamationIVT). Samples were analyzed by SDS-PAGEand Western blot, or by MesoScale Discovery (MSD) Plate Assay. Protocolsfor these assays are described elsewhere in the Materials and Methods(p-r). A standard curve was generated for the MSD Plate Assay using 6C1.

In the resulting Western blots using either the 9D5 or 5A1 mis-TTR mAb,differences between normal and TTR-V30M diseased plasma samples wereevident. All plasma samples contained an ˜14 kDa TTR band thatco-migrated with the non-native TTR monomer present in the pH4-TTRreference sample. In general, plasma samples derived from TTR-V30Mpatients (#21, 22, 23, 24, 25, & 27) had more of this mis-TTR species.In addition, plasma samples derived from V30M patients also contained an˜30 kDa band that co-migrates with the non-native TTR dimer present inthe reference sample. With the exception of samples #12 and #18, plasmasamples derived from normal individuals possessed less of this dimerspecies.

The resulting Western blots were scanned and the intensities of thecombined 9D5- or 5A1-reactive TTR dimer and monomer bands were plottedfor each sample (the results are shown in FIG. 5A (9D5) and 5B (5A1) andare presented in arbitrary units (a.u.) on the y-axis). With theexception of plasma samples #15 and #18, plasma samples derived fromnormal individuals (11, 12, 19, and 20) contained less 9D5 reactivedimer and monomer than samples derived from V30M patients (21-25 and27).

The 12 serum samples analyzed by 9D5 and 5A1 Western blot were alsoanalyzed by MSD plate assay using 6C1 as the mis-TTR capture antibodyand the Dako-SulfoTag antibody as the detection antibody. Results ofthese MSD assays are shown in FIG. 6 and are presented in arbitraryunits (a.u.) on the y-axis. Samples 11, 12, 15, 18, 19, and 20 representnormal plasma. Samples 21-25 and 27 represent V30M diseased plasma.

With the exception of plasma samples #15 and #18, the amount of6C1-reactive TTR present in plasma samples derived from normalindividuals was lower than that observed in plasma from TTR-V30Mdiseased individuals. The levels of 6C1 reactivity measured by MSD assaycorrelated very well with the amount of 9D5 reactive dimer and monomerobserved above by SDS-PAGE/Western.

In order to determine the concentration of the reactive TTR speciespresent in plasma samples, the same samples were re-assayed using 6C1 asthe capture antibody and 8C3-SulfoTag as the detection antibody. MSDsignals were converted to ng/ml concentrations of reactive TTR speciesusing the TTR F87M/L110M standard curve generated above. Based on thisanalysis, the average concentration of 6C1-reactive TTR present in thecontrol samples was 271+/−185 ng/ml. In contrast, the averageconcentration of reactive TTR present in the V30M diseased plasmasamples was higher, at 331+/−95 ng/ml. Taken together, these MSD resultssuggest that mis-TTR antibodies are capable of distinguishing betweenATTR disease versus normal plasma. This warrants further development ofmis-TTR antibodies for use in diagnostic assays of ATTR disease.

Example 7. Design of Humanized 6C1 Antibodies

The starting point or donor antibody for humanization was the mouseantibody 6C1. The heavy chain variable amino acid sequence of maturem6C1 is provided as SEQ ID NO:1. The light chain variable amino acidsequence of mature m6C1 is provided as SEQ ID NO:13. The heavy chainCDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ IDNOS:10-12, respectively. The light chain CDR1, CDR2, and CDR3 amino acidsequences are provided as SEQ ID NOS:18-20, respectively. Kabatnumbering is used throughout in this Example.

The variable kappa (Vk) of m6C1 belongs to mouse Kabat subgroup 2, whichcorresponds to human Kabat subgroup 2. The variable heavy (Vh) of m6C1belongs to mouse Kabat subgroup 3d, which corresponds to Kabat subgroup3. See Kabat et al. Sequences of Proteins of Immunological Interest,Fifth Edition. NIH Publication No. 91-3242, 1991. The 16-residue CDR-L1belongs to canonical class 4, the 7-residue CDR-L2 belongs to canonicalclass 1, and the 9-residue CDR-L3 belongs to canonical class 1 in Vk.See Martin & Thornton, J. Mol. Biol. 263:800-15, 1996. The 10-residueCDR-H1 (a composite of Chothia and Kabat CDR-H1, residues 26-35 as shownin FIGS. 12A-12D) belongs to canonical class 1, and the 17-residueCDR-H2 belongs to canonical class 1. See Martin & Thornton, J Mol. Biol.263:800-15, 1996. The CDR-H3 has no canonical classes.

The residues at the interface between the Vk and Vh domains are the onescommonly found.

A search was made over the protein sequences in the PDB database(Deshpande et al., Nucleic Acids Res. 33: D233-7, 2005) to findstructures which would provide a rough structural model of 6C1. Thecrystal structure of antibody fab (pdb code 3EYS) (Gardberg et al.,Biochemistry (2009) Vol. 48(23), pp. 5210-5217) was used for Vkstructure since it had good resolution (1.95 A), overall sequencesimilarity to 6C1 Vk, and retained the same canonical structure for theloops as 6C1. A dimeric antibody (pdb code 2OTU) (Li et al., Submissionto GenBank (2007)) was used for the Vh structure since it had goodsimilarity and resolution (1.68 A) and contained the same canonicalstructures for CDR-H1 and CDR-H2 as that of 6C1 VH. BioLuminate software(licensed from Schrodinger Inc.) was used to model a rough structure of6C1.

A search of the non-redundant protein sequence database from NCBIallowed selection of suitable human frameworks into which to graft themurine CDRs. For Vh, human Ig heavy chain ADX65650 (GI: 323432015) (SEQID NO:3) was chosen (Scheel et al., Submission to GenBank (2010)). Itshares the canonical forms of 6C1. For Vk, a human kappa light chainwith NCBI accession code ABI74084 (GI: 114385652) was chosen (SEQ IDNO:15) (Shriner et al. Submission to GenBank (2006)). It has the samecanonical classes for CDR-L1 and L2 as that for the parental Vk.

Six humanized heavy chain variable region variants and two humanizedlight chain variable region variants were constructed containingdifferent permutations of substitutions (Hu6C1VHv1, Hu6C1VHv1b,Hu6C1VHv2, Hu6C1VHv2b, Hu6C1VHv3, and Hu6C1VHv3b (SEQ ID NOS:4-9,respectively) and Hu6C1VLv1-2 (SEQ ID NOS:16 and 17, respectively))(FIGS. 12A-12D and FIGS. 13A-13D). The exemplary humanized Vh and Vkdesigns, with backmutations and other mutations based on selected humanframeworks, are shown in FIGS. 12A-12D and FIGS. 13A-13, respectively.The gray-shaded areas in the first column in FIGS. 12A-12D and FIGS.13A-13D indicate the CDRs as defined by Chothia, and the gray-shadedareas in the remaining columns in FIGS. 12A-12D and FIGS. 13A-13Dindicate the CDRs as defined by Kabat. SEQ ID NOS:4-9, 16, and 17contain backmutations and other mutations as shown in Table 7. The aminoacids at positions L2, L45, H19, H44, H49, H76, H77, H82(a), H83, andH89 in Hu6C1VHv1, Hu6C1VHv1b, Hu6C1VHv2, Hu6C1VHv2b, Hu6C1VHv3, andHu6C1VHv3b, and in Hu6C1VLv1-2, are listed in Table 8.

TABLE 7 V_(H), V_(L) Backmutations and Other Mutations V_(H) or V_(L)Exon Donor Framework V_(H) or V_(L) Variant Acceptor Sequence ResiduesHu6C1VHv1 NCBI accession H77 (SEQ ID NO: 4) code ADX65650 (SEQ ID NO: 3)Hu6C1VHv1b NCBI accession H49, H77 (SEQ ID NO: 5) code ADX65650 (SEQ IDNO: 3) Hu6C1VHv2 NCBI accession H76, H77, H82(a) (SEQ ID NO: 6) codeADX65650 (SEQ ID NO: 3) Hu6C1VHv2b NCBI accession H49, H76, H77, H82(a)(SEQ ID NO: 7) code ADX65650 (SEQ ID NO: 3) Hu6C1VHv3 NCBI accessionH19, H44, H77, H83, H89 (SEQ ID NO: 8) code ADX65650 (SEQ ID NO: 3)Hu6C1VHv3b NCBI accession H19, H44, H49, H77, H83, H89 (SEQ ID NO: 9)code ADX65650 (SEQ ID NO: 3) Hu6C1VLv1 NCBI accession L2, L45 (SEQ IDNO: 16) code ABI74084 (SEQ ID NO: 15) Hu6C1VLv2 NCBI accession L45 (SEQID NO: 17) code ABI74084 (SEQ ID NO: 15)

TABLE 8 Kabat Numbering of Framework Residuesfor Backmutations and Other Mutations in Humanized 6C1 AntibodiesADX65650 ABI74084 Heavy Light Mouse Hu6C1 Hu6C1 Hu6C1 Hu6C1 Hu6C1 Hu6C1Hu6C1 Hu6C1 Residue Chain Chain 6C1 VHv1 VHv1b VHv2 VHv2b VHv3 VHv3bVLv1 VLv2 L2 — I V — — — — — — V I L45 — Q K — — — — — — K K H19 R — K RR R R K K — — H44 G — R G G G G R R — — H49 S — A S A S A S A — — H76 N— N N N S S N N — — H77 S — T T T T T T T — — H82(a) N — S N N S S N N —— H83 R — K R R R R K K — — H89 V — M V V V V M M — —

An alignment of the murine 6C1 Vh sequence (SEQ ID NO:1) with the mousemodel sequence (2 OUT_B.pro; SEQ ID NO:2), the human acceptor sequence(ADX65650; SEQ ID NO:3), and the Hu6C1VHv1, Hu6C1VHv1b, Hu6C1VHv2,Hu6C1VHv2b, Hu6C1VHv3, and Hu6C1VHv3b sequences (SEQ ID NOS:4-9,respectively), is shown in FIG. 1. The CDR regions as defined by Kabatare shaded. Positions at which canonical, vernier, or interface residuesdiffer between mouse and human acceptor sequences are candidates forsubstitution. Examples of vernier/CDR foundation residues include Kabatresidues 2, 49, 69, 71, 75, 78, and 94 in FIGS. 12A-12D. Examples ofcanonical/CDR interacting residues include Kabat residues 24, 48, and 73in FIGS. 12A-12D. Examples of interface/packing (VH+VL) residues includeKabat residues 37, 39, 45, 47, 91, 93, and 103 in FIGS. 12A-12D.

An alignment of the murine 6C1 Vk sequence (SEQ ID NO:13) with the mousemodel sequence (3EYS_L_St.pro; SEQ ID NO:14), the human acceptorsequence (ABI74084; SEQ ID NO:15), and the Hu6C1VLv1 and Hu6C1VLv2sequences (SEQ ID NOS:16 and 17, respectively), is shown in FIG. 2. TheCDR regions as defined by Kabat are shaded. Positions at whichcanonical, vernier, or interface residues differ between mouse and humanacceptor sequences are candidates for substitution. Examples ofvernier/CDR foundation residues include Kabat residues 4, 35, 46, 49,66, 68, and 69 in FIGS. 13A-13D. Examples of canonical/CDR interactingresidues include Kabat residues 2, 48, 64, and 71 in Table 8. Examplesof interface/packing (VH+VL) residues include Kabat residues 36, 38, 44,87, and 98 in FIGS. 13A-13D.

The rationales for selection of the positions indicated in Tables 7 and8 in the light chain variable region as candidates for substitution areas follows.

I2V: This is a canonical CDR interacting residue. The bulkier side chainof Ile could potentially interfere with CDRs L1 and L2 packing. Thisresidue is backmutated to Val in Hu6C1VH1.

Q45K: Lys is more frequent at this position than Gln in the humansequence; therefore this is a frequency based backmutation.

The rationales for selection of the positions indicated in Tables 9 and10 in the heavy chain variable region as candidates for substitution areas follows.

R19K: Lys forms H-bonds with adjoining residues whereas Arg does not.

G44R: Arg forms H-bonds with interface residue Phe98 in the light chain,whereas Gly does not.

S49A: Ser can potentially form an H-bond with Hys in CDR-H2.

N76S: There is a high deamidation exposure at this residue. Ser issecond most frequent in human germline at this position.

S77T: Serine at this position is very rare in the human germane heavychain frameworks, whereas, Threonine is most frequent at position 77.This back mutation has been made to mitigate any immunogenecitypotential.

N82(a)S: There is a high deamidation exposure at this residue. Ser issecond most frequent in human germline at this position.

R83K: Lys at this position forms multiple interactions with adjoiningresidues, seeming to exert a stabilizing effect on the loop, whereas Argdoes not.

V89M: Met forms H-bonds with interference residue Tyr91 and appears tostabilize the interface, whereas Val does not interact with Tyr91.

The two humanized light chain variable region variants and two humanizedheavy chain variable region variants are as follows:

Hu6C1VL version 1 (I2V and Q45K backmutations in lowercase):(SEQ ID NO: 16) DvVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPkWYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLT FGGGTKVEIKHu6C1VL version 2 (Q45K backmutation in lowercase): (SEQ ID NO: 17)DIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPkWYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLT FGGGTKVEIKHu6C1VH version 1 (S77T backmutation in lowercase): (SEQ ID NO: 4)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVSYISIDGNNIYHPDSVKGRFTISRDNAKNtLYLQMNSLRAEDTAVYYCARDS DYGYFDVWGQGTLVTVSHu6C1VH version 1b (S49A and S77T backmutations in lowercase):(SEQ ID NO: 5) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVaYISIDGNNIYHPDSVKGRFTISRDNAKNtLYLQMNSLRAEDTAVYYCARDS DYGYFDVWGQGTLVTVSSHu6C1VH version 2 (N76S, S77T, and N82(a)S backmutations in lowercase):(SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVSYISIDGNNIYHPDSVKGRFTISRDNAKstLYLQMsSLRAEDTAVYYCARDS DYGYFDVWGQGTLVTVSSHu6C1VH version 2b (S49A, N765, S77T, and N82(a)Sbackmutations in lowercase): (SEQ ID NO: 7)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVaYISIDGNNIYHPDSVKGRFTISRDNAKstLYLQMsSLRAEDTAVYYCARDS DYGYFDVWGQGTLVTVSSHu6C1VH version 3 (R19K, G44R, S77T, R83K, andV89M backmutations in lowercase): (SEQ ID NO: 8)EVQLVESGGGLVQPGGSLkLSCAASGFTFSNYYMSWVRQAPGKrLEWVSYISIDGNNIYHPDSVKGRFTISRDNAKNtLYLQMNSLkAEDTAmYYCARDS DYGYFDVWGQGTLVTVSS Hu6C1VH version 3b (R19K, G44R, S49A, S77T, R83K,and V89M backmutations in lowercase): (SEQ ID NO: 9)EVQLVESGGGLVQPGGSLkLSCAASGFTFSNYYMSWVRQAPGKrLEWVaYISIDGNNIYHPDSVKGRFTISRDNAKNtLYLQMNSLkAEDTAmYYCARDS DYGYFDVWGQGTLVTVSS

Example 8. Binding Kinetic Analysis of Humanized 6C1 Antibodies

Binding kinetics of humanized 6C1 antibodies comprising a heavy chainselected from version 3b and a light chain selected from version 2 werecharacterized by Biacore and shown below.

mAb k_(a)(1/Ms) k_(d)(1/s) K_(D)(M) R_(max) Hu-6C1-H3bL2 3.724E+55.449E−4 1.463E−9 38.80

Example 9. Materials and Methods

a. Antibody Generation Protocol

Mice were immunized weekly with the antigenic peptides TTR-MAP,TTR89-97-N-KLH or TTR89-97-C-KLH in RIBI adjuvant or monthly in TiterMaxadjuvant. Three to four days prior to fusion, selected mice were given afinal IV boost with immunogen in saline solution. Spleen werehomogenized to prepare splenocytes and fused with SP2/0 myeloma cellsusing a standard electrofusion protocol. Fused cells in selection mediawere plated in 96-well plates and screened after 7-10 days.

b. Antibody Screening Protocol

Hybridoma selection was based on the following ELISA screen: 96-wellELISA plates were coated with chicken anti-His, 1 μg/mL PBS andincubated for 1 hour. Plates were blocked with of 1% BSA/PBS solution,200 uL/well for 15 minutes then 0.5 μg/mL pH4-TTR, 50 μL/well was addedand incubated for 1 hour. pH4-TTR is TTR that has been subjected to lowpH (50 mM sodium acetate, pH 4.0) in order to dissociate/aggregate TTR,exposing the TTR89-97 epitope. Plates were washed twice with TBS-T.Supernatant from fusion plates was added, 50 μL/well and incubated for 1hour. Plates were washed twice with TBS-T. The detection antibody, goatanti-mouse (IgG1, 2a, 2b, 3 specific)-HRP diluted 1:5,000 in 0.5%BSA/PBS/TBS-T, 50 μL/well was added and incubated for 1 hour. Finally,plates were washed five times with TBS-T and TMB substrate, 100 μL/wellwas added. After 15 minutes, substrate development was stopped with 2NSulfuric Acid, 50 μL/well. Plates were read at 450 nm. Wells with anO.D.>1.0 were selected and cells were transferred to a 24-well plate.After 3 days of growth, clones were counter screened with the aboveassay to confirm binding, and substituting native TTR for pH4-TTR as anegative counter screen, allowing for selection of clones producing TTRmAbs specific for non-native forms of TTR.

c. Antibody Expression Protocols

CMV driven light chain and heavy chain plasmids carrying humanizedmonoclonal antibody sequences were transfected into CHO-S1 cells (LifeTechnology). Dual selection was applied to make a selected pool.Conditioned media was assayed for titer, binding and analyzed bySDS-PAGE/Western blotting. Selected pools were used for clone generationusing Clonepix system (Molecular Devices). Clones were ranked based onantibody titer. Selected clones were expanded and banked.

The highest producing clone was expanded in shake flasks and the culturewas used to inoculate 10-25 L Wave bag cultures. A mixture ofFreeStyle-CHO, CD OptiCHO and FreeStyle F17 expression mediasupplemented with Glutamax (media and Glutamax from Life Technology) wasused for shake flask as well as for Wave bag cultures. Batch culture wasmade using a Wave Bioreactor (GE Heathcare) at 37° C., 7% CO₂ underconstant agitation. Samples were drawn periodically to monitor cellnumber, viability and antibody production. Supplementation with CellBoost (HyClone) was made if needed. The batch culture was harvested whencell viability starts to decline below 90% (5-7 days).

d. Antibody Purification Protocol

The cell culture was harvested after first allowing the cells insuspension to settle down to the bottom of the Wave bag via gravity at4° C. Harvested media was clarified through a depth filter (MillistakPod COHC, Millipore), concentrated 10-fold by tangential flow filtration(Pelicon 2PLC 30K, Millipore) and sterile filtered through a 0.2 μmfilter (Opticap XL, Millipore). The concentrated conditioned media wasthen loaded onto a Protein G Sepharose Fast Flow column (GELifesciences) pre-equilibrated in 1×PBS, pH 7.4 using an FPLC (AktaAvant, GE Lifesciences). Unbound proteins were washed off the columnwith 5-10 column volumes of 1×PBS, pH 7.4 until the OD₂₈₀ reachedbaseline. The bound antibody was eluted from the column with 2 columnvolumes of IgG Elution Buffer (Thermo Scientific). Elution fractionswere collected and pH neutralized with 2M Tris, pH 9.0 (60 μL per 1 mlelution).

Antibody-containing fractions were pooled and dialyzed overnight at 4°C. against 1×PBS, pH 7.4. The dialyzed sample was then sterilized byultrafiltration through a 0.2 μm PES filter and stored at 4° C. Thefinal protein concentration was determined by bicinchoninic acid (BCA)using bovine gamma-globulin as the protein standard (Thermo Scientific).

e. Recombinant TTR Expression and Purification Protocols

E. coli (BL21-A1) cells were transformed with a pET21a(+) plasmidcontaining a TTR insert (Met-hTTR-(His)₆ or a TTR variant containing anF87M/L110M double mutation. Cells were grown in 2YT broth containing 100μg/ml ampicillin. Expression of TTR was induced overnight at 20° C. inthe presence of 1 mM IPTG and 005% arabinose.

The cells were collected by centrifugation at 4000×g for 10 min. andstored at −80° C. until used. 10-15 g cell pellets were thawed and lysedin 50m1 Buffer A (1×PBS containing 500 mM NaCl, 20 mM imidazole) byprocessing through an LV-1 high-shear processor (Microfluidics, Inc.).Lysed cells were centrifuged at 12,000×g for 15 min, filtered through a0.2 μm PES filter prior to purification on a His-Trap HP column (GELifesciences). After loading, the column was washed with 10 c.v. ofBuffer A and eluted with Buffer B (1×PBS with 500 mM NaCl, 500 mMimidazole). Peak fractions corresponding to TTR were collected, dialyzedagainst 1×PBS and stored at −80° C. until used.

f. TTR Antigen Preparation

Native TTR antigen was prepared by diluting a concentrated stock ofrecombinant TTR-6His to a final concentration of 2.5 μg/ml in 1×PBS.pH4-treated TTR was generated by incubating recombinant TTR at aconcentration of 0.2 mg/ml in 50 mM sodium acetate, pH 3.95 for 72 hoursat room temperature. Under these conditions, TTR dissociates intomixture of TTR monomers and aggregated forms that are structurallydistinct from native TTR. The pH4-TTR was then diluted to a finalconcentration of 2.5 μg/ml in 1×PBS immediately before use in the assay.96-well plates (Costar #3690) were coated at room temperature with 50 μlper well of 1.0 μg/ml chicken-anti-his polyclonal antibody (Abcam#Ab9107) in 1×PBS for 1 hr. The coating solution was discarded and theplate was blocked with a 250 μl/well volume of 1×BSA-containing blockbuffer diluted in 1×PBS (G-Biosciences #786-193) for 1 hr.

g. ELISA Protocol

Coated and blocked 96-well plates were treated with 50 μl per well of2.5 μg/ml TTR antigen (either native TTR or pH4-TTR) for 1 hr. at roomtemperature. The plates were then washed two times with 250 μl per wellof wash buffer (1× Tris Buffered Saline containing 0.05% Tween-20).Washed plates were then treated with 50 μl per well of the appropriateanti-TTR monoclonal antibody at concentrations ranging from of 0.31 to2.5 μg/ml, for 1 hr.

The treated plates were washed 3 times with 250 μl perwell wash buffer.After washing, the plates were treated for 1 hr. with 50 μl per well ofdetection antibody comprising a 1:5,000 dilution of peroxide-conjugatedgoat-anti-mouse (Jackson ImmunoResearch #115-035-164) in 1×PBS. Theplate was then washed 3 times prior to the addition of 100 μl per wellTMB substrate (Rockland). The HRP reaction was allowed to proceed atroom temperature for 15 min. before quenching with a 50 μl per wellvolume of 1N H2504. Spectroscopic absorbance was measured at awavelength of 450 nm.

h. SDS-PAGE

Electrophoresis on SDS-polyacrylamide gels was carried out as follows.0.1-1 μg TTR or pH 4.0-TTR in 1×LDS sample buffer (Life Technologies)was loaded onto a 10% NuPAGE bis-tris gel and subjected toelectrophoresis in IVIES buffer at a constant 90V for 105 minutes. Afterelectrophoresis, the gel was either stained in Instant Blue (Expedeon)or transferred to nitrocellulose filters for Western blot analysis.

i. Native-PAGE

Electrophoresis on native Tris-glycine gels was carried out as follows.0.1-1 μg TTR or pH 4.0-TTR in 1×Tris-glycine sample buffer (LifeTechnologies) was loaded onto a 10-20% Tris-glycine gel and subjected toelectrophoresis in 1× Native Tris-glycine running buffer at a constant120V for 105 minutes. After electrophoresis, the gel was either stainedin Instant Blue (Expedeon) or transferred to nitrocellulose filters forWestern blot analysis.

Western Blot

SDS- or Native-PAGE gels were blotted onto nitrocellulose filter paper(iBlot, P7 Program) and blocked with blocking buffer (Licor) for 30minutes. The filters were then incubated in 0.5 μg/ml primary antibodyin blocking buffer for 1 hour at room temperature (or over-night at 4°C.), followed by three, 10 minutes washes with 1×TBS. The filters wereplaced in IRDye 800CW-conjugated goat-anti-mouse secondary diluted1:20,000 in block buffer. After incubating the filters in secondaryantibody solution for 1 hour at room temperature, the filters werewashed and imaged on an Odyssey CLx infrared imager (Licor).

k. TTR Fiber Formation Assay Procotol

A solution of 3.6 μM (0.2 mg/ml) TTR-Y78F in 50 mM sodium acetate, pH4.8 was incubated at 37° C. for 72 hours in the presence of 1.4 μM (0.2mg/ml) mis-TTR antibody or an isotype control. After incubation, a 5×molar excess of thioflavin-T was added to the mixture and allowed tobind for 30 minutes. Fluorometric measurements were measured at anemissions wavelength of 480 nm with an excitation wavelength set at 440nm. The 0% inhibition was set as the fluorescence intensity in thepresence of an isotype control antibody (83 a.u.) and the 100%inhibition point was set as the fluorescence in the absence of TTR-Y78Fprotein (38 a.u.).

1. Cardiac Tissue Samples

Fresh frozen and paraffin-processed blocks of cardiac tissue withconfirmed diagnoses of ATTR mutations were obtained from Dr. MerrillBenson at Indiana University. Samples included eight fresh frozensamples and six FFPE samples and each sample was diagnosed with eitherATTR or some other cardiac amyloidosis. The diagnosis of the tissue wasfurther confirmed at Prothena via IHC staining with antibodies to kappaand lambda light chains and amyloid A prior to characterization with theTTR antibodies.

m. Immunohistochemistry

Immunohistochemistry was performed on lightly paraformaldheyde-fixed, 10μm slide-mounted cryosections and on 5 μm paraffin sections. Theimmunoperoxidase method was the principal detection system, which wasperformed on the Leica Bond Rx (Leica Biosystems, Buffalo Grove, Ill.)using the Bond Polymer Refine Detection Kit (DS980, Leica Biosystems).The primary antibodies were incubated for one hour (according toconcentrations in Table 2.) followed by incubation with anti-mouse andanti-rabbit polymeric HRP-linker antibody conjugates. The staining wasvisualized with a DAB chromogen, which produced a brown deposit. Theslides were counterstained with hematoxylin, dehydrated in an ascendingseries of alcohols, cleared in xylenes, and coverslipped with CytoSeal60 (Richard Allen Scientific; Kalamazoo, Mich.). Negative controlconsisted of performing the entire immunohistochemical procedure onadjacent sections with a non-immune IgG isotype control or an omissionof the primary antibody.

n. Demonstration of Amyloid: Congo Red and Thioflavin T Staining

Congo red stain was performed to demonstrate TTR amyloid in the tissueusing a kit from American MasterTech (Lodi, Calif.). The staining wasperformed according to the manufacturer's procedure. Slides were stainedin the Congo Red solution for 1 hour followed by differentiation in 1%sodium hydroxide for approximately 15 seconds. The slides were thenrinsed in running water, dehydrated through an alcohol series ofincreasing concentrations, and cleared through three changes of xylenes,and coverslipped with CytoSeal 60.

A modified Thioflavin T staining protocol (Schmidt et al 1995.) wasemployed to determine the presence of TTR amyloid in the tissue.Briefly, slides were counterstained with a Mayers hematoxylin, rinsed inrunning water and stained with a filtered solution of 0.015% ThioflavinT (T3516-25G; Sigma-Aldrich, St. Louis, Mo.) in 50% ethanol for tenminutes. The slides were then rinsed in running water and differentiatedin 1% (v/v) acetic acid for 10 minutes and rinsed three times in water.The slides were allowed to air dry before being coverslipped withProLong Gold (Life Technologies).

o. Image Analysis

Slides were imaged with either an Olympus BX61 microscope, HamamatsuNanozoomer 2.0HT digital slide scanner, or a Leica SPE spectral confocalsystem. Images were collected and stored as TIFF files.

p. Analysis of Human Plasma Samples by SDS-PAGE/Western

Six plasma samples from patients confirmed for V30M ATTR (Sample #11,#12, #15, #18, #19, #20) and 6 samples (#21, #22, #23, #24, #25, #27)from normal subjects were obtained from M. Saraiva (Porto University,Portugal). Sample #C6 was a normal human serum sample obtained from acommercial source (BioreclamationIVT). These plasma samples wereseparated by SDS-PAGE and Western blotted with 9D5 as follow. A 1.4 μlvolume of plasma was diluted 1:8 into 1×LDS sample buffer in the absenceof reducing agent (Life Technologies). Samples were subjected toSDS-PAGE separation and Western blotted with 0.5 μg/ml 9D5 as describedpreviously.

q. Analysis of Human Plasma Samples by MesoScale Discovery (MSD) PlateAssay

96-well MSD plates were coated with monoclonal antibody 6C1 at aconcentration of 4 μg/mL in PBS and incubated for 2 hours at roomtemperature with shaking, or overnight at 4° C. Plates were washed threetimes with 1×TBST before being blocked with of 3% MSD Blocker Asolution, 150 μL per well for 1 hour shaking. A 30 μl per well volume ofhuman plasma samples diluted 1:10 in a sample buffer comprised of 0.6%globulin-free bovine serum albumin, 1.5 mM monobasic sodium phosphate, 8mM dibasic sodium phosphate, 145 mM sodium chloride, 0.05% Triton X-405,and 0.05% thimerosal was added to the blocked MSD plates for 1 hour.Plates were washed 3 times with 1×TBST. A 50 μl per well volume of 1μg/ml sulfo-tagged detection antibody (either 8C3 total TTR antibody ofthe Dako polyclonal antibody) in sample buffer was added for 1 hr. atroom temperature with shaking. Plates were washed three times with1×TBST followed by the addition of 150 μl per well 1× Read Buffer Tsolution (Meso Scale Discovery). Plates were then read in the MSD Sectorimager.

r. Generation of an MSD Standard Curve

In order to quantitate the amount of non-native, 6C1-reactive TTRprotein present in human plasma samples, a MSD standard curve wasgenerated using recombinant TTR-F87M/L110M as a 6C1-reactive TTRstandard. This TTR variant contains two amino acid substitutions thatprevent tetramer formation and keeps the protein in the monomer state(Jiang et al. (2001) Biochemistry 40, 11442-11452). As such, this TTRvariant is recognized by all mis-TTR mAbs and is therefore well-suitedfor use as a reference standard in the MSD assay.

To generate the standard curve, 96-well MSD plates were coated withmis-TTR antibody 6C1 at a concentration of 4 μg/mL in PBS and incubatedfor 2 hours at room temperature with shaking, or overnight at 4° C.Plates were washed three times with 1×TBST before being blocked with of3% MSD Blocker A solution, 150 μL per well for 1 hour shaking. Theblocked plates were then treated for 1 hour with 50 μl per well of 25μg/mL TTR-F87M/L110M serially diluted 1:5 with the last dilution being abuffer blank. Plates were washed 3 times with 1×TBST before the additionof a 50 μl per well volume of 1 μg/ml SulfoTag-detection antibody(8C3-SulfoTag or Dako pAb-SulfoTag) for 1 hour at room temperature withshaking. Both 8C3 mAb and the Dako antibody were coupled to the SulfoTagand could be used at the detection antibody since they bound to totalTTR and were not conformation specific.

After treatment with the detection antibody, plates were washed threetimes with a 150 μl per well volume of 1×TBST, followed by the additionof 150 μl per well 1× Read Buffer T (MSD). Plates were read in the MSDSector imager and a resulting TTR F87M/L110M calibration curve wasgenerated.

Example 10. Evaluation of Mis-TTR. Antibodies in Transgenic Louse Model

In vivo studies are conducted in a humanized transgenic mouse model V30MhTTR (Inoue et al., (2008) Specific pathogen free conditions preventtransthyretin amyloidosis in mouse models. Transgenic Research17:817-826) to assess the efficacy of anti-TTR antibodies in the bindingand removal of aggregated hTTR.

Transgenic mice are bred using standard procedures and their circulatinghTTR levels are assessed by ELISA. Mice with a serum level of 200-400μg/ml of hTTR are used for subsequent efficacy studies. The first set ofstudies examine the natural deposition of hTTR in transgenic mice.Detection of hTTR deposits begins at 12 months of age and is repeatedevery 3-6 months thereafter. Once an acceptable level of aggregates isseen in transgenic mice, efficacy studies are initiated. Animals aredivided into three treatment groups (n=10/group) and treated weekly forfour weeks with an IP dose of vehicle, control antibody (isotypecontrol, 10 mpk) or an anti-hTRR antibody (10 mpk). One week after thelast treatment the mice are euthanized, tissues collected and processed,and then stained to assess the number and size of remaining TTRdeposits. Quantitative methods and statistics are employed to determinethe degree of clearance seen among groups.

In an alternative approach, hTTR aggregates are prepared in vitro andthen injected into the kidney of transgenic mice to seed the depositionof new aggregates. Applicant has determined that the injection of thesepreparations can expedite the deposition of new aggregates in apredictable manner. Based on these findings, animals are sedated, theleft kidney exposed and pre-aggregated hTTR material injected into thecortex of the kidney. After a suitable recovery period, mice are dividedinto three treatment groups (n=10/group) and treated weekly forfour-eight weeks with an IP dose of vehicle, control antibody (isotypecontrol, 10 mpk) or an anti-hTRR antibody (10 mpk). One week after thelast treatment the mice are euthanized, the kidneys collected andprocessed, and then stained to assess the number and size of TTRdeposits. Quantitative methods and statistics are employed to determinethe degree of change seen among groups.

Example 11. Evaluation of Mis-TTR Antibodies in a Matrigel Implant Model

Applicant has determined that pre-aggregated hTTR can be suspended inMatrigel (BD Bioscience, Cat #354263), allowed to solidify and thenplaced subcutaneously in mice. At four weeks post implantation, theMatrigel implant maintained its structure and the aggregated hTTR wasstill present within the implant. Moreover, the implant was welltolerated by the mice and anti-hTTR antibodies were able to penetrateand bind to the aggregates suspended in the Matrigel. Based on thesefindings, an antibody efficacy study is conducted. Animals are sedatedand an implant containing pre-aggregated hTTR suspended in Matrigelplaced subcutaneously in mice. After a suitable recovery period, miceare divided into three treatment groups (n=10/group) and treated weekly,for two-four weeks with an IP dose of vehicle, control antibody (isotypecontrol, 10 mpk) or an anti-hTRR antibody (10 mpk). After the lasttreatment, the mice are euthanized, the skin containing the implantcollected and processed, and then the amount of TTR deposits remainingassessed using histological and/or biochemical methods. Quantitativeanalysis and statistics are employed to determine the degree ofclearance seen among groups.

Example 12 Electron and Atomic Force Microscopy

Immunogold transmission electron microscopy (TEM) and atomic forcemicroscopy (AFM) were used to generate images of the interaction betweenmis-TTR mAbs and both aggregated and fibrillar forms of the protein.Isothermal titration calorimetry (ITC) was carried out by titrating 14G8into a solution of aggregated TTR using standard methods. Using TEM,immunogold labeling with 14G8 was observed in TTR-V122I oligomeraggregates and fibril ends (FIG. 8A), whereas immunogold labeling withan anti-TTR pAb shows binding along the lengths of TTR fibers and tooligomeric clusters (FIG. 8B). IgG1 isotype control mAb does not showimmunogold labeling (FIG. 8C). TTR-V122I fibers, alone and in thepresence of 14G8±6 nm colloidal gold-conjugated secondary antibody, wereassessed using AFM. Gold labeling was observed at fiber ends (FIG. 8D).FIG. 9A shows isothermal titration calorimetry (ITC) data and bindingisotherms for 14G8 binding to aggregated TTR variants. FIG. 9B showsbinding fitted to a 2-binding site model with KD values shown. TEM, AFM,and ITC analysis provide evidence mis-TTR mAbs bind to TTR aggregatesand fibrils primarily at 2 distinct sites: oligomers and fibril ends.

Example 13: Antibody Binding to TTR Amyloid in the Peripheral Nerves andGastrointestinal Tract of a Patient with ATTR Amyloidosis from aTTR-V30M Mutation

14G8 and control antibodies were evaluated immunohistochemically todetermine their reactivity for TTR amyloid deposits in nerve, andgastrointestinal tract samples obtained from patients with confirmeddiagnoses of ATTR amyloidosis from a V30M mutation. FIGS. 10A-G show14G8 immunolabeled TTR amyloid present between fibers of the nervefascicle (FIG. 10A panels 1 and 2), which overlapped with staining byCongo red (FIG. 10B panels 1 and 2) and thioflavin T (FIG. 10C panels 1and 2), and immunolabeling by a total-TTR antibody (FIG. 10D) in tissuederived from a patient with ATTR amyloidosis. No staining was seen withthe use of 2 isotype control antibodies (FIGS. 10E-F); however, axonaldegeneration (lack of Schwann cell nuclei) in the areas laden with TTRamyloid deposits were also observed (FIGS. 10E-F [red areas in 10E]).Peripheral nerves from a healthy control were not labeled using either14G8 or a total-TTR antibody (FIG. 10G panels 1-3)

14G8 labelled TTR amyloid deposited throughout the gastrointestinaltract; Meissner's plexus and glands in the esophagus (FIG. 11A, B panels1), the rich vasculature bed in the submucosa (FIG. 11C panel 1), andthe muscularis propria (MP) and muscularis mucosa (MM) of the jejunum(FIG. 11D panel 1) were immunolabeled with 14G8. 14G8-positive TTRamyloid overlapped with Congo red fluorescent staining (FIGS. 11A-Dpanels 2). ATTR amyloidosis tissue stained with an isotype control mAb(FIGS. 119A-D panels 3). 14G8 immunoreactivity was absent in healthycontrol tissue (FIG. 11E panels 1-4)

These findings provide evidence that mis-TTR mAbs can be useful inpreventing the deposition or enhancing the clearance, or both, of TTRamyloid in patients with ATTR amyloidosis regardless of the specificorgan(s) involved while sparing the function of the normal tetramericform of the protein.

1-67. (canceled)
 68. The method of claim 73, wherein administering theantibody inhibits or reduces aggregation of transthyretin in thesubject.
 69. The method of claim 73, wherein administering the antibodyinhibits or reduces transthyretin fibril formation in the subject. 70.The method of claim 73, wherein administering the antibody reducestransthyretin deposits in a subject.
 71. The method of claim 73, whereinadministering the antibody clears aggregated transthyretin in thesubject relative to a subject having or at risk of developing atransthyretin-mediated amyloidosis who has not received the antibody.72. The method of claim 73, wherein administering the antibodystabilizes a non-toxic conformation of transthyretin in the subject. 73.A method of treating or effecting prophylaxis of atransthyretin-mediated amyloidosis in a subject, comprisingadministering to the subject an effective regime of an antibody thatspecifically binds transthyretin comprising three heavy chain CDRs andthree light chain CDRs substantially from antibody 6C1.
 74. The methodof claim 73, wherein administering the antibody delays the onset of atransthyretin-mediated amyloidosis in the subject
 75. The method ofclaim 73, wherein the transthyretin-mediated amyloidosis is associatedwith a condition selected from any of cardiomyopathy or hypertrophy,familial amyloid polyneuropathy, central nervous system selectiveamyloidosis (CNSA), senile systemic amyloidosis, senile cardiacamyloidosis, spinal stenosis, osteoarthritis, rheumatoid arthritis,juvenile idiopathic arthritis, age related macular degeneration, and aligament or tendon disorder.
 76. A method of treating a subject havingor at risk of any of cardiomyopathy or hypertrophy, familial amyloidpolyneuropathy, central nervous system selective amyloidosis (CNSA),senile systemic amyloidosis, senile cardiac amyloidosis, spinalstenosis, osteoarthritis, rheumatoid arthritis, juvenile idiopathicarthritis, age related macular degeneration, and a ligament or tendondisorder, the method comprising administering to the subject aneffective regime of the antibody of an antibody that specifically bindstransthyretin comprising three heavy chain CDRs and three light chainCDRs substantially from antibody 6C1. 77-94. (canceled)
 95. The methodof claim 73, wherein the antibody comprises three Kabat heavy chain CDRs(SEQ ID NOS: 10-12, respectively) and three light CDRs (SEQ ID NOS:18-20, respectively).
 96. The method of claim 95, wherein the antibodyhas a human IgG1 isotype.
 97. The method of claim 73, wherein theantibody is a humanized or chimeric 6C1 antibody that specifically bindsto transthyretin, wherein 6C1 is a mouse antibody characterized by amature heavy chain variable region of SEQ ID NO:1 and a mature lightchain variable region of SEQ ID NO:13.
 98. The method of claim 73,wherein the antibody comprises a humanized mature heavy chain variableregion having an amino acid sequence at least 90% identical to SEQ IDNO:9 and a humanized mature light chain variable region having an aminoacid sequence at least 90% identical to SEQ ID NO:17.
 99. The method ofclaim 95, provided position H77 of the antibody is occupied by T. 100.The method of claim 99, provided position H49 of the antibody isoccupied by A.
 101. The method of claim 99, provided positions H76 andH82(a) of the antibody are occupied by S.
 102. The method of claim 99,provided positions H19, H44, H83, and H89 of the antibody are occupiedby K, R, K, and M, respectively.
 103. The method of claim 95, providedposition L45 of the antibody is occupied by K.
 104. The method of claim103, provided position L2 of the antibody is occupied by V.
 105. Themethod of claim 98, wherein the antibody comprises a mature heavy chainvariable region having an amino acid sequence at least 95% identical toSEQ ID NO:9 and a mature light chain variable region having an aminoacid sequence at least 95% identical to SEQ ID NO:17.
 106. The method ofclaim 105, wherein the antibody comprises a mature heavy chain variableregion having an amino acid sequence at least 98% identical to SEQ IDNO:9 and a mature light chain variable region having an amino acidsequence at least 98% identical to SEQ ID NO:17.
 107. The method ofclaim 97, wherein the mature heavy chain variable region of the antibodyhas the amino acid sequence of SEQ ID NO:4.
 108. The method of claim 97,wherein the mature heavy chain variable region of the antibody has theamino acid sequence of SEQ ID NO:5.
 109. The method of claim 97, whereinthe mature heavy chain variable region of the antibody has the aminoacid sequence of SEQ ID NO:6.
 110. The method of claim 97, wherein themature heavy chain variable region of the antibody has the amino acidsequence of SEQ ID NO:7.
 111. The method of claim 97, wherein the matureheavy chain variable region of the antibody has the amino acid sequenceof SEQ ID NO:8.
 112. The method of claim 97, wherein the mature heavychain variable region of the antibody has the amino acid sequence of SEQID NO:9.
 113. The method of any one of claims 107-112, wherein themature light chain variable region of the antibody has the amino acidsequence of SEQ ID NO:16 or SEQ ID NO:17.
 114. The method of claim 73,wherein the mature heavy chain has the amino acid sequence of SEQ ID NO:9 and the mature light chain variable region of the antibody has anamino acid sequence of SEQ ID NO:17.
 115. The method of claim 73 or 106,wherein the antibody is a binding fragment.
 116. The method of claim115, wherein the binding fragment is a single-chain antibody, Fab, orFab′2 fragment.
 117. The method of claim 73 or 106, wherein the maturelight chain variable region of the antibody is fused to a light chainconstant region and the mature heavy chain variable region of theantibody is fused to a heavy chain constant region.
 118. The method ofclaim 117, wherein the heavy chain constant region is a mutant form of anatural human heavy chain constant region which has reduced binding to aFcγ receptor relative to the natural human heavy chain constant region.119. The method of claim 117, wherein the mature heavy chain variableregion is fused to a heavy chain constant region having the sequence ofSEQ ID NO:26 and/or the mature light chain variable region is fused to alight chain constant region having the sequence of SEQ ID NO:28.